Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion.

The distribution and phylogeny of Wolbachia in filarial species suggests that these endosymbiotic bacteria may be important in the biology of their filarial hosts. An experiment to falsify this hypothesis would be to treat filarial worms with antibiotics which are active against intracellular bacteria. Indeed, it has already been shown that tetracycline treatment inhibits development in a model filarial species (Brugia pahangi) at different stages of the life cycle, in both mosquito and mammalian hosts. Here we discuss these previous data and present new results on the effect of tetracycline on the embryogenesis of the canine filaria Dirofilaria immitis.     

A new insight into the pathogenesis of filarial disease

Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.     

Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.     

The filarial endosymbiont Wolbachia sp. is absent from Setaria equina

Wolbachia sp. was first reported in filarial nematodes over 25 yr ago. Today, much research is focused on the role of these bacteria in filarial worm biology. The filarial symbionts are closely related to arthropod symbionts, which are known to modify host reproduction and biology through various mechanisms. Similarly, it has been suggested that Wolbachia sp. is essential for long-term survival and reproduction of filariae. We report that Wolbachia sp. 16S rDNA was not found in the equine filarial nematode Setaria equina, using either polymerase chain reaction (PCR) or DNA hybridization. In addition, ultrastructural analysis of adult worms did not reveal the presence of Wolbachia sp. in hypodermal cords or reproductive tissues. These data suggest that like Onchocerca flexuosa and Acanthocheilonema vitae, S. equina may not be dependent on Wolbachia sp. for survival.     

Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis

Filarial nematodes harbour intracellular endosymbiotic bacteria, which have been assigned to the genus Wolbachia. These bacteria appear to play an important role in the pathogenesis of filarial diseases through their lipopolysaccharides. In view of the presence of Wolbachia endosymbionts in the body of filarial nematodes, one might also expect that proteins from these bacteria play an antigenic role in humans and animals affected by filariases. To test this hypothesis, we produced in recombinant form the surface protein WSP and a portion of the cell-cycle protein FTSZ from the Wolbachia of Dirofilaria immitis. Western immunoblot assays were then performed using cat sera to test the immunogenicity of these proteins. Sera were collected from owners' cats, which were either sero-negative or sero-positive for D. immitis and from cats before and after experimental infection with D. immitis. FTSZ was recognized in Western blots by sera from both positive and negative cats and from both uninfected and experimentally infected cats. WSP was recognized only by sera from positive cats and from cats experimentally infected with D. immitis; this protein was not recognized by sera from negative cats and from cats before experimental infection with D. immitis. The results of Western blot assays on WSP thus support the hypothesis that infection with filarial nematodes induces the production of antibodies against Wolbachia proteins.     

Are filarial nematode Wolbachia obligate mutualist symbionts?

The intracellular symbiotic bacteria of filarial nematodes have inspired new ideas for the control of disease using antibacterial drugs. For effective, long-term control, this requires that the bacteria are essential to their nematode hosts. Two recent studies offer conflicting evidence: long, close coevolution between most filarial nematodes and their symbionts contrasts with many species having naturally lost them. An attempt to transfer symbionts to an uninfected host found that the bacteria did not thrive, suggesting they are adapted to one host.     

Phylogeny of Wolbachia in filarial nematodes.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.     

Litomosoides sigmodontis: vaccine-induced immune responses against Wolbachia surface protein can enhance the survival of filarial nematodes during primary infection

Wolbachia are bacteria present within the tissues of most filarial nematodes. Filarial nematode survival is known to be affected by immune responses generated during filarial nematode infection and immune responses to Wolbachia can be found in different species harbouring filarial nematode infections, including humans. Using the rodent filarial model Litomosoides sigmodontis, we show that pre-exposure to wolbachia surface protein in a Th1 context (but not in a Th2-context) enhances worm survival on subsequent challenge. This study suggests that despite abundant evidence that pro-inflammatory reactions to the endosymbiont have detrimental effects on the both the nematode and mammalian host, they may under some circumstances be beneficial to the nematode.     

Sequencing of the complete gene coding for the GroEL of the Wolbachia of Dirofilaria immitis and expression and purification of the recombinant protein

Wolbachia are intracellular bacteria that infect arthropods and filarial nematodes. These bacteria play an important role in the immunology and pathogenesis of filarial diseases through their proteins and, possibly, other molecules. GroEL is a constitutively expressed bacterial protein; it is highly conserved among bacteria and is involved in the correct folding of newly synthesized proteins. Here we report the production of recombinant GroEL from the Wolbachia of Dirofilaria immitis. Our goal is to test the hypothesis that GroEL is involved in the immunopathology of filariases. The complete groel gene was PCR-amplified, sequenced and cloned into an expression vector. The recombinant GroEL was purified by affinity chromatography by using high-performance liquid chromatography (HPLC).     

Real-time PCR for quantification of the bacterial endosymbionts (Wolbachia) of filarial nematodes

Filarial nematodes harbour intracellular symbiotic bacteria belonging to the genus Wolbachia. Wolbachia is thought to play an important role in the biology of the nematode. Moreover, Wolbachia appears to be involved in the immunopathogenesis of filariasis and in the onset of the side-effects of antifilarial therapy. Investigations in these research areas require reliable methods to quantify Wolbachia both in nematodes and in vertebrate tissues. To this purpose, we designed a quantitative real-time PCR targeted on the ftsZ gene of the Wolbachia of Brugia pahangi, a model filarial species maintained in gerbils. The method was applied to quantify Wolbachia in Brugia pahangi, from animals with or without tetracycline treatment. Our results show that tetracycline treatment leads to dramatic reduction or clearance of Wolbachia from the nematode. Results obtained from different replicates were reproducible and the method appeared very sensitive compared to other PCR protocols for Wolbachia detection. Real-time PCR is thus an appropriate method for investigations on the biological role of Wolbachia and on the implication of these bacteria in the pathogenesis of filariasis. With slight modifications of the primers and probe, the protocol we have developed could be applied in studies of the human pathogen Brugia malayi and on the model filarial species Litomosoides sigmodontis.     

Ultrastructural evidence of the degenerative events occurring during embryogenesis of the filarial nematode Brugia pahangi after tetracycline treatment

Intracellular bacteria belonging to the genus Wolbachia have been described in filarial nematodes and these microorganisms appear to have evolved an obligatory mutualistic association with their filarial hosts. In fact, antibiotic treatment leads to the clearance of bacteria from worms resulting in a block in embryogenesis and, eventually, death of adult filariae. Currently, the antifilarial action of antibiotic treatment is interpreted as a secondary consequence of the bacteriostatic activity against Wolbachia endosymbionts. Here, we demonstrate by transmission electron microscopy the degenerative events occurring during embryogenesis of Brugia pahangi after tetracycline treatment. After 56 days of treatment the cytoplasm of hypodermal cords was totally void of Wolbachia and numerous vacuoles, residual of cytolitic activity, were observed. In the ovary, the morphology of the oocytes was well conserved 33 days after treatment, but the texture of symbiotic bacteria appeared altered. After 56 days of treatment embryogenesis was dramatically affected and the terminal portion of the ovary appeared totally empty. The authors suggest that the symbiotic bacteria play a direct role in worm metabolism and a long-term bacteriostatic effect may block bacterial activity involved in the active control of cytolysis. As a consequence, the bacteriophorous vacuole is transformed into a digestive vacuole and the whole symbiotic population is disrupted.     

Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites

In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.     

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.     

Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM

The discovery that endosymbiotic Wolbachia bacteria play an important role in the pathophysiology of diseases caused by filarial nematodes, including lymphatic filariasis and onchocerciasis (river blindness) has transformed our approach to these disabling diseases. Because these parasites infect hundreds of millions of individuals worldwide, understanding host factors involved in the pathogenesis of filarial-induced diseases is paramount. However, the role of early innate responses to filarial and Wolbachia ligands in the development of filarial diseases has not been fully elucidated. To determine the role of TLRs, we used cell lines transfected with human TLRs and macrophages from TLR and adaptor molecule-deficient mice and evaluated macrophage recruitment in vivo. Extracts of Brugia malayi and Onchocerca volvulus, which contain Wolbachia, directly stimulated human embryonic kidney cells expressing TLR2, but not TLR3 or TLR4. Wolbachia containing filarial extracts stimulated cytokine production in macrophages from C57BL/6 and TLR4(-/-) mice, but not from TLR2(-/-) or TLR6(-/-) mice. Similarly, macrophages from mice deficient in adaptor molecules Toll/IL-1R domain-containing adaptor-inducing IFN-beta and Toll/IL-1R domain-containing adaptor-inducing IFN-beta-related adaptor molecule produced equivalent cytokines as wild-type cells, whereas responses were absent in macrophages from MyD88(-/-) and Toll/IL-1R domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) deficient mice. Isolated Wolbachia bacteria demonstrated similar TLR and adaptor molecule requirements. In vivo, macrophage migration to the cornea in response to filarial extracts containing Wolbachia was dependent on TLR2 but not TLR4. These results establish that the innate inflammatory pathways activated by endosymbiotic Wolbachia in B. malayi and O. volvulus filaria are dependent on TLR2-TLR6 interactions and are mediated by adaptor molecules MyD88 and TIRAP/Mal.     

Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.     

The eye does not see what the mind does not know: the bacterium in the worm

Symbiotic relationships underlie the evolutionary success of many different life forms. The filarial worms are long, slender nematode parasites that cause considerable pathology in large segments of the world's population. About 25 years ago, investigators first reported the presence of bacterial organisms living inside these parasitic worms. Recent molecular biological studies have indicated that these bacteria belong to the genus Wolbachia, members of which have been known to be associated with numerous species of insects. Elimination of the Wolbachia from the nematodes (by, for instance, treatment with broad spectrum antibiotics) results in profound disturbances in the physiology of the latter, including a complete block in reproduction. This observation, taken together with the fact that every individual worm examined to date contains Wolbachia, validates the classification of the latter as "endosymbionts." Many studies indicate that the Wolbachia may also play an important role in the pathology caused by the nematode worms, and that it might be possible to target therapy against the Wolbachia to treat the filarial disease. Intriguingly, the intense recent interest in Wolbachia is in complete contrast with the virtually complete indifference evoked by the original discovery of these organisms.     

Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria <Emphasis Type="Italic">Wolbachia</Emphasis>

Background  

Outer membrane proteins (OMPs) of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP) can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods.     

Clarithromycin and Amoxicillin Susceptibility of Helicobacter pylori Strains Isolated from Adult Patients with Gastric or Duodenal Ulcer in Italy

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences. Received: 10 January 2000 / Accepted: 22 February 2000     

Endosymbiotic Wolbachia bacteria as biological control tools of disease vectors and pests

Wolbachia bacteria are common cytoplasmic symbionts of insects, mites and filarial nematodes. They can alter the reproduction of their hosts. The symbiont could be eliminated, transferred or used through genetic alteration to take advantage or remove their possible influences on pests and/or natural enemies. Their extensive effects on reproduction and host fitness have made Wolbachia the subject of growing attention as a potential biocontrol agent. Here, we summarize the relations of Wolbachia in the control of disease vectors and pests. Furthermore, the drawbacks of these bacteria are also discussed.