Claudio Genchi

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 to 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.

Heartworm Disease in Animals and Humans

Heartworm disease due to Dirofilaria immitis continues to cause severe disease and even death in dogs and other animals in many parts of the world, even though safe, highly effective and convenient preventatives have been available for the past two decades. Moreover, the parasite and vector mosquitoes continue to spread into areas where they have not been reported previously. Heartworm societies have been established in the USA and Japan and the First European Dirofilaria Days (FEDD) Conference was held in Zagreb, Croatia, in February of 2007. These organizations promote awareness, encourage research and provide updated guidelines for the diagnosis, treatment and prevention of heartworm disease. The chapter begins with a review of the biology and life cycle of the parasite. It continues with the prevalence and distribution of the disease in domestic and wild animals, with emphasis on more recent data on the spreading of the disease and the use of molecular biology techniques in vector studies. The section on pathogenesis and immunology also includes a discussion of the current knowledge of the potential role of the Wolbachia endosymbiont in inflammatory and immune responses to D. immitis infection, diagnostic use of specific immune responses to the bacteria, immunomodulatory activity and antibiotic treatment of infected animals. Canine, feline and ferret heartworm disease are updated with regard to the clinical presentation, diagnosis, prevention, therapy and management of the disease, with special emphasis on the recently described Heartworm Associated Respiratory Disease (HARD) Syndrome in cats. The section devoted to heartworm infection in humans also includes notes on other epizootic filariae, particularly D. repens in humans in Europe. The chapter concludes with a discussion on emerging strategies in heartworm treatment and control, highlighting the potential role of tetracycline antibiotics in adulticidal therapy.

A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts

Infection with the endosymbiotic bacteria Wolbachia is widespread in filarial nematodes. Previous studies have suggested concordance between the phylogeny of Wolbachia with that of their nematode hosts. However, there is only one published molecular phylogenetic study of filarial species, based on the 5S rRNA gene spacer. The phylogeny proposed by this study is partially incongruent with previous classifications of filarial nematodes, based on morphological characters. Furthermore, both traditional classifications and molecular phylogenies are, in part, inconsistent with the phylogeny of Wolbachia. Here we report mitochondrial cytochrome oxidase I (COI) gene sequences for 11 species of filaria and for another spirurid nematode which was included as an outgroup. In addition, 16S rRNA, wsp and ftsZ gene sequences were generated for the Wolbachia of several filarial species, in order to complete the available data sets and further resolve the phylogeny of Wolbachia in nematodes. We used these data to evaluate whether nematode and Wolbachia phylogenies are concordant. Some of the possible phylogenetic reconstructions based on COI gene were congruent with the phylogeny of Wolbachia and supported the grouping of the rodent filaria Litomosoides sigmodontis with the lymphatic filariae (i.e. Brugia spp. and Wuchereria spp.) and the sister group relationship of Dirofilaria spp. and Onchocerca spp. However, the placement of the Wolbachia-free filaria Acanthocheilonema viteae is ambiguous and dependent on the phylogenetic methods used.

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 northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors

Visceral leishmaniasis (VL) incidence has been increased in Italy in humans and dogs since the 1990s, with new foci being detected within traditional boundaries of endemic transmission but also in northern regions previously regarded as non‐endemic. To monitor the putative VL spreading, surveillance was implemented in northern continental Italy comprising: analysis of human cases recorded from 1990 through 2005; retrospective literature analysis of canine leishmaniasis (CanL) and phlebotomine sandfly records through 2002; prospective investigations in dogs from 2003 through 2005 and surveys on sandflies in 2003 and 2004. Two‐hundred‐thirty human cases (11% of Italian cases) were recorded. Their stratification by age and HIV status disclosed a sharp decrease of HIV/VL co‐infections paralleled by concomitant increase of paediatric and HIV‐negative adult patients during the study period. Four patients had no travel history. Seven leishmaniasis foci were retrospectively identified since 1990, whereas prospective investigations in dogs disclosed 47 autochthonous clinical cases and 106 autochthonous seropositives among 5442 dogs (2.1%) from 16 foci of six regions. Parasites were typed as Leishmania infantum MON‐1. Four vector species were identified among 1696 Phlebotomus (Larroussius) collected specimens. Comparisons with historical data showed that P. perniciosus and P. neglectus have increased in density and expanded their geographic range in the study area. Northern continental Italy is now focally endemic for VL and a moderate risk for human disease does exist, although the intensity of transmission seems to be lower than in traditional settings of Mediterranean VL.

Climate and Dirofilaria infection in Europe

Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of several vector borne parasites like Dirofilaria, and in the risk of infection for animals and humans. The present paper reviews the effects of climate and other global drivers on Dirofilaria immitis and Dirofilaria repens infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes. In the last several years, growing degree day (GDD)-based forecast models, which use wide or local scale temperature data, have been developed to predict the occurrence and seasonality of Dirofilaria in different parts of the world. All these models are based on the fact that: there is a threshold of 14 degrees C below which Dirofilaria development will not proceed; and there is a requirement of 130 GDD for larvae to reach infectivity and a maximum life expectancy of 30 days for a vector mosquito. The output of these models predicts that the summer temperatures (with peaks in July) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. These factors not only favour incubation of Dirofilaria, but also impact on mosquito species. Recent findings have also demonstrated that Aedes albopictus is now considered to be an important, competent vector of Dirofilaria infections. This mosquito species could spread from southern to northern European countries in the near future, changing the epidemiological patterns of dirofilariosis both in humans and animals.

Vector-borne helminths of dogs and humans in Europe

Presently, 45% of the total human population of Europe, as well as their domestic and companion animals, are exposed to the risk of vector-borne helminths (VBH) causing diseases. A plethora of intrinsic biological and extrinsic factors affect the relationship among helminths, vectors and animal hosts, in a constantly changing environment. Although canine dirofilarioses by Dirofilaria immitis and Dirofilaria repens are key examples of the success of VBH spreading into non-endemic areas, another example is represented by Thelazia callipaeda eyeworm, an emergent pathogen of dogs, cats and humans in several regions of Europe. The recent finding of Onchocerca lupi causing canine and human infestation in Europe and overseas renders the picture of VBH even more complicated. Similarly, tick-transmitted filarioids of the genus Cercopithifilaria infesting the skin of dogs were recently shown to be widespread in Europe. Although for most of the VBH above there is an increasing accumulation of research data on their distribution at national level, the overall impact of the diseases they cause in dogs and humans is not fully recognised in many aspects. This review investigates the reasons underlying the increasing trend in distribution of VBH in Europe and discusses the diagnostic and control strategies currently available. In addition, this article provides the authors’ opinion on some topics related to VBH that would deserve further scientific investigation.

Dirofilarial infections in Europe.

Nematodes of the genus Dirofilaria are currently considered emerging agents of parasitic zoonoses in Europe. Climatic changes and an increase in the movement of reservoirs (mostly infected dogs) have caused an increase in the geographical range of these parasites from the traditionally endemic/hyperendemic southern regions, and the risk for human infection has increased. In the last several years, forecast models have predicted that current summer temperatures are sufficient to facilitate extrinsic incubation of Dirofilaria in many areas of Europe. The global warming projected by the Intergovernmental Panel on Climate Change suggests that warm summers suitable for Dirofilaria transmission in Europe will be the rule in the future decades, and if the actual trend of temperature increase continues, filarial infection should spread into previously infection-free areas. Dirofilaria repens is currently the filarial species that is most commonly reported as spreading from southern to northern areas. This article reviews the zoonotic aspects, effects of climate, and other global drivers on Dirofilaria infections in Europe and the possible implications on the transmission and control of these mosquito-borne nematodes.

Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6 microg/kg per os weekly) combined with doxycycline (DOXY; 10 mg/kg/day orally from Weeks 0-6, 10-12, 16-18, 22-26 and 28-34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.

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.