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.     

First record of the bacterial endosymbiont Wolbachia for phytophagous hoverflies from genus Merodon (Diptera: Syrphidae)

Wolbachia is a widespread bacterial endosymbiont among arthropod species. It influences the reproduction of the host species and also mitochondrial DNA diversity. Until now there were only a few studies that detected Wolbachia infections in hoverflies (Diptera: Syrphidae), and this is the first broader study with the aim of examining the incidence of Wolbachia in the hoverfly genus Merodon. The obtained results indicate an infection rate of 96% and the presence of both Wolbachia supergroup A and B, which are characteristic for most of the infected arthropod species. Additionally, the presence of multiple Wolbachia strains in the Merodon aureus group species was detected and the mitochondrial DNA COI‐based relationships of the group are discussed in the light of infection. Finally, we discuss plant‐mediated horizontal transmission of Wolbachia strains among the studied hoverfly species.     

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).     

Immunisation of mice with fractions derived from the intestines of Dirofilaria immitis

Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.     

Two cases of ectopic dirofilariasis by Dirofilaria immitis in subconjunctival and subcutaneous tissues in dogs

We report two cases of ectopic dirofilariasis caused by Dirofilaria immitis in the subconjunctival and subcutaneous tissues of dogs. In Case 1, a 10-year-old female poodle suffered from a subconjunctival mass in the left eye that was refractory to antibiotic and steroid treatments. The mass was removed surgically, and a whitish nematode was identified within the mass. In Case 2, a stray 3-year-old male greyhound was rescued and transferred to a local veterinary hospital. During care, two nematodes were observed on the left hind paw and leg. The observed worms were not typical of D. immitis; therefore, the species was confirmed using molecular methods. Phylogenetic analysis revealed high genetic identity with other previously reported D. immitis strain. Subcutaneous and subconjunctival dirofilariasis have been mainly caused by D. repens. To the best of our knowledge, this is the first report of subconjunctival ectopic dirofilariasis by D. immitis in a dog, and the first report of subcutaneous localization in Korea. Therefore, in endemic regions, ectopic dirofilariasis caused by D. immitis should be considered as a differential diagnosis in subconjunctival and subcutaneous masses.     

Dirofilaria immitis: biochemical and immunological characterization of the surface antigens from adult parasites

The molecules associated with the surface of adult Dirofilaria immitis were identified and characterized employing IODO-GEN-mediated surface labeling methods. D. immitis female and male parasites were found to have a limited number of surface-associated proteins (17.5, 16, and 14.5 kDa) and glycoproteins (49 and 20 kDa) which were readily extracted from parasite homogenates in the absence of detergent. The major surface labeled proteins and glycoproteins were antigenic in rabbits, but appeared to elicit only a weak humoral response in dogs with patent dirofilariasis. In addition, a 10- to 6-kDa surface-associated glycolipid was identified which may form a coat on the outside of the parasite and play a role in immune evasion. In immunoprecipitation experiments, the glycolipid was not recognized by the antibodies from rabbits exposed to the glycolipid or by antibodies in the sera of patently infected animals. The glycolipid and the 14.5-kDa surface protein were selectively released by the adult parasite during in vitro culture.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Phosphofructokinase from Dirofilaria immitis. Stimulation of activity by phosphorylation with cyclic AMP-dependent protein kinase

Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.     

Dirofilaria immitis: experimental infections in the ferret (Mustela putorius furo)

The ferret, Mustela putorius furo, was found to be susceptible to Dirofilaria immitis infection when exposed to low (14) or high (280-420) numbers of infective larvae harvested from Aedes aegypti. Eight ferrets (half of them cortisonized) were inoculated subcutaneously with 14 larvae each. All of them were subsequently found to harbor D. immitis in the heart, and all but one of them had worms of both sexes. Six of these ferrets were examined for microfilaremia at 31 to 35 weeks after inoculation; 3 were positive (one observed only at postmortem examination) and there was evidence that fertilization of female worms had occurred in one other. Females up to 25.5 cm and males up to 16.0 cm were recovered. There was no evidence that the cortisonization of some ferrets had affected the infections. Both male and female ferrets became infected. Four cortisonized ferrets were inoculated with 280 or 420 larvae of D. immitis (divided equally between subcutaneous and intraperitoneal routes). All of them died 16 to 18 weeks after inoculation, yielding 102 to 125 immature D. immitis. In these lethal infections, worms were recovered from the heart and adjoining vessels, and also from vascular and extravascular sites throughout the body.     

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.     

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

Summary

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.     

Biochemical characterization of Recombinase A from Wolbachia endosymbiont of filarial nematode Brugia malayi (wBmRecA)

Lymphatic filariasis is a debilitating disease that affects over 890 million people in 49 countries. A lack of vaccines, non-availability of adulticidal drugs, the threat of emerging drug resistance against available chemotherapeutics and an incomplete understanding of the immunobiology of the disease have sustained the problem. Characterization of Wolbachia proteins, the bacterial endosymbiont which helps in the growth and development of filarial worms, regulates fecundity in female worms and mediates immunopathogenesis of Lymphatic Filariasis, is an important approach to gain insights into the immunopathogenesis of the disease. In this study, we carried out extensive biochemical characterization of Recombinase A from Wolbachia of the filarial nematode Brugia malayi (wBmRecA) using an Electrophoretic Mobility Shift Assay, an ATP binding and hydrolysis assay, DNA strand exchange reactions, DAPI displacement assay and confocal microscopy, and evaluated anti-filarial activity of RecA inhibitors. Confocal studies showed that wBmRecA was expressed and localised within B. malayi microfilariae (Mf) and uteri and lateral chord of adult females. Recombinant wBmRecA was biochemically active and showed intrinsic binding capacity towards both single-stranded DNA and double-stranded DNA that were enhanced by ATP, suggesting ATP-induced cooperativity. wBmRecA promoted ATP hydrolysis and DNA strand exchange reactions in a concentration-dependent manner, and its binding to DNA was sensitive to temperature, pH and salt concentration. Importantly, the anti-parasitic drug Suramin, and Phthalocyanine tetrasulfonate (PcTs)-based inhibitors Fe-PcTs and 3,4-Cu-PcTs, inhibited wBmRecA activity and affected the motility and viability of Mf. The addition of Doxycycline further enhanced microfilaricidal activity of wBmRecA, suggesting potential synergism. Taken together, the omnipresence of wBmRecA in B. malayi life stages and the potent microfilaricidal activity of RecA inhibitors suggest an important role of wBmRecA in filarial pathogenesis.     

Identification of immunoreactive proteins of Dirofilaria immitis and D. repens recognized by sera from patients with pulmonary and subcutaneous dirofilariosis

Human pulmonary and subcutaneous dirofilariosis caused by Dirofilaria immitis and Dirofilaria repens are worldwide diagnosed with increasing frequency. These species are responsible for the development of benign pulmonary and subcutaneous nodules, respectively, that can be confused with lung or cutaneous cancer. The aim of the present work was to identify D. immitis and D. repens proteins differentially recognized by serum samples from individuals with human pulmonary and subcutaneous dirofilariosis, using two-dimensional electrophoresis and mass spectrometry. Twenty-three immunoreactive proteins of D. immitis and 15 of D. repens were identified. The results point to the existence of differential antigenic recognition in each species, both in the number and type of proteins recognized. Individuals with pulmonary dirofilariosis recognized, on the proteome of D. immitis, among others, different isoforms of 6 enzymes involved in glycolysis, 3 redox-related proteins with antioxidant capacity and 3 heat shock proteins. Individuals with subcutaneous dirofilariosis recognized on the proteome of D. repens only 3 glycolytic enzymes, one protein involved in redox processes and one heat shock protein. These data suggest that in cases of pulmonary dirofilariosis there exists a wider recognition of immunoreactive D. immitis proteins related to key survival processes, such as energy generation, the struggle against oxidative stress and molecular repair, than in cases of human subcutaneous dirofilariosis against D. repens. This could contribute to explain the differences described in the capacity of D. immitis and D. repens development and in the frequency of occurrence of pulmonary and subcutaneous dirofilariosis in the human host.     

Further characterization of refractoriness in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy)

Factors which control the expression of the refractory or susceptible condition to infection with Dirofilaria immitis in the mosquito. Aedes aegypti, were investigated using three protocols. (1) Microfilariae and prelarvae were injected into the hemocoel of susceptible A. aegypti. Some microfilariae and prelarvae developed to the L1 larval stage but they failed to complete development to the infective stage. (2) Enema of microfilariae and prelarvae from infected susceptible and refractory donor females were given into the midgut of uninfected susceptible and refractory recipient females. The results indicate that the conditions which inhibit the initiation of development are present in the Malpighian tubules and not in the midgut of the refractory mosquitoes. (3) Transplants of infected Malpighian tubules from susceptible and refractory donor females were made into the abdominal hemocoel of uninfected susceptible and refractory recipient females. The results showed that the refractory condition depends on the genetic makeup of the donor, not the recipient, mosquito. The above results taken as a whole indicate that the factors which control refractoriness are not present in the midgut but are present in the Malpighian tubule cells of refractory A. aegypti.     

Molecular detection of Wolbachia pipientis in natural populations of mosquito vectors of Dirofilaria immitis from continental Portugal: first detection in Culex theileri

Wolbachia pipientis (Rickettsiales: Rickettsiaceae) protects mosquitoes from infections with arboviruses and parasites. However, the effect of its co‐infection on vector competence for Dirofilaria immitis (Spirurida: Onchocercidae) in the wild has not been investigated. This study aimed to screen vectors of D. immitis for wPip, to characterize these, and to investigate a possible association between the occurrence of W. pipientis and that of the nematode. The presence of W. pipientis was assessed in the five mosquito potential vectors of D. immitis in Portugal. Polymerase chain reaction (PCR) products were sequenced, and wPip haplotypes were determined by PCR–restricted fragment length polymorphism (RFLP). Results showed that wPip was detected in 61.5% of Culex pipiens (Diptera: Culicidae) pools and 6.3% of Culex theileri pools. wPip 16s rRNA sequences found in Cx. theileri exactly match those from Cx. pipiens, confirming a mosquito origin, rather than a nematode origin, as some specimens were infected with D. immitis. Only wPip haplotype I was found. No association was found between the presence of wPip and D. immitis in mosquitoes and hence a role for this endosymbiont in influencing vectorial competence is yet to be identified. This study contributes to understanding of wPip distribution in mosquito populations and, to the best of the authors' knowledge, is the first report of natural infections by wPip in Cx. theileri.     

Efficacy of moxidectin for the prevention of adult heartworm (Dirofilaria immitis) infection in dogs

The authors report the efficacy of orally administered moxidectin for the prevention of canine heartworm infection in two endemic areas in northern Italy. Two trials were conducted on a total of 257 dogs, including 137 treated with moxidectin (minimum dose of 3 mcg/kg body weight), 85 with ivermectin (minimum dose 6.6 mcg/kg b.w.) and 35 untreated controls. Results of testing for microfilariae and circulating adult female antigens were negative for all treated dogs at the end of both trials. No adverse reactions to moxidectin were observed. In the study areas, prevalence values for Dirofilaria immitis infection calculated on the basis of the untreated controls and testing dogs which had no preventive treatment in the previous transmission season ranged 23-65%. This study confirms the efficacy and safety of moxidectin in the prevention of adult heartworm infection in dogs.     

温度通过影响Wolbachia滴度调控赤眼蜂生殖方式

【目的】揭示温度对宿主体内Wolbachia滴度及其调控宿主生殖作用的影响。【方法】以感染Wolbachia且营孤雌产雌生殖的食胚赤眼蜂Trichogramma embryophagum为对象,在22,25,28和31℃4个梯度温度下连续培养5代,观察生殖方式、性比等生物学特性;采用实时荧光定量PCR以Wolbachia特异基因——外膜蛋白基因(wsp)、二磷酸果糖醛缩酶基因(fbp A)和酰胺转移酶基因(gat B)为靶标对内生菌进行定量分析。【结果】22和25℃条件下连续培养5代,食胚赤眼蜂生殖方式均未发生改变(无雄蜂出现),且5代赤眼蜂群体Wolbachia滴度没有明显差异;28℃下,食胚赤眼蜂在F3代开始有雄蜂出现,至F5代雄蜂比例明显增多,且体内Wolbachia滴度在F2代开始下降,至F5代显著下降;31℃下,F2代即有雄蜂出现,至F5代已经恢复成产雄孤雌生殖,体内Wolbachia滴度也在F2代开始下降,F3代开始显著降低,到F5代Wolbachia滴度极小甚至检测不到。【结论】高温可改变营孤雌产雌生殖赤眼蜂的生殖方式及体内Wolbachia滴度,且随着处理代数的增加以及温度的升高作用显著,即高温对营孤雌产雌生殖赤眼蜂生殖方式改变程度与其体内Wolbachia滴度呈负相关。