Search

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.

The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as 'paratransgenesis', has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

Background: Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals., Methodology/principal Findings: We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected., Conclusions/significance: The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.

The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Recent research in microbe-insect symbiosis has shown that acetic acid bacteria (AAB) establish symbiotic relationships with several insects of the orders Diptera, Hymenoptera, Hemiptera, and Homoptera, all relying on sugar-based diets, such as nectars, fruit sugars, or phloem sap. To date, the fruit flies Drosophila melanogaster and Bactrocera oleae, mosquitoes of the genera Anopheles and Aedes, the honey bee Apis mellifera, the leafhopper Scaphoideus titanus, and the mealybug Saccharicoccus sacchari have been found to be associated with the bacterial genera Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, and Saccharibacter and the novel genus Commensalibacter. AAB establish symbiotic associations with the insect midgut, a niche characterized by the availability of diet-derived carbohydrates and oxygen and by an acidic pH, selective factors that support AAB growth. AAB have been shown to actively colonize different insect tissues and organs, such as the epithelia of male and female reproductive organs, the Malpighian tubules, and the salivary glands. This complex topology of the symbiosis indicates that AAB possess the keys for passing through body barriers, allowing them to migrate to different organs of the host. Recently, AAB involvement in the regulation of innate immune system homeostasis of Drosophila has been shown, indicating a functional role in host survival. All of these lines of evidence indicate that AAB can play different roles in insect biology, not being restricted to the feeding habit of the host. The close association of AAB and their insect hosts has been confirmed by the demonstration of multiple modes of transmission between individuals and to their progeny that include vertical and horizontal transmission routes, comprising a venereal one. Taken together, the data indicate that AAB represent novel secondary symbionts of insects.

Wolbachia is the most widespread bacterial endosymbiont in insects. It is responsible for a variety of reproductive alterations of the hosts. Wolbachia is transmitted through the germline from mother to offspring and, in rare cases, between individuals. This implies that acquired properties (through symbiosis with Wolbachia) can become heritable. We investigated the transovarial inheritance of Wolbachia in two phylogenetically distant insects, Drosophila melanogaster and Zyginidia pullula. We detected in both systems bacteriocyte-like cells, densely packed with Wolbachia endosymbionts, at the tip of the ovarioles. Bacteriocytes are cells specialized to harbour bacteria, typical of mutualistic insect symbiosis. Our observations of bacteriocyte-like cells harbouring Wolbachia in the ovary emphasize the plasticity of the female reproductive system of insects, which maintains its function while some cells are densely colonized by bacteria. In summary, there is evidence from different insects that bacteria which behave as parasites of reproduction are harboured by cells resembling bacteriocytes, which appear to mediate transmission of the bacteria to the progeny. It seems a valid hypothesis that the bacteriocyte-like cells that we observed are not the result of a co-evolution of host and symbiont, considering that Wolbachia is not an obligatory symbiont in Drosophila and Zyginidia., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

We aimed to determine the presence of Ixodes ricinusticks in heavily populated areas of the Po River Valley after report of a Lyme disease case. Eighteen percent of ticks examined from 3 locations were positive for Lyme disease borreliae. Lyme disease was diagnosed for 3 workers at risk for tick bite.

Amanita phalloides, Lepiota cristata, Lepiota brunneoincarnata and Inocybe asterospora are among the most important species responsible of mushroom poisoning in northern Italy. A real time PCR method for the identification of samples containing DNA from each of these species was developed. To test specificity all protocols were applied on DNA extracted from various mushroom species; sensitivity was assessed performing serial dilutions on all samples; versatility of the protocols was evaluated performing tests on DNA extracted from different matrices. The protocols showed high sensitivity (32 ng dried mushroom), high specificity and sensitive detection of DNA extracted from difficult samples, including pasta with mushroom, cooked mushrooms and gastric aspirates.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E(2). These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

Anisakids are nematodes whose larval stages are often present in fish, molluscs, and crustaceans. Members of the family Anisakidae belonging to the genera Anisakis and Pseudoterranova are implicated in human infections caused by the consumption of raw or undercooked fish. Adequate cooking will kill anisakid larvae, however, killed or inactivated larvae can still cause sensitization and immunoglobulin E-dependent hypersensitivity in human. This work describes the development of DNA-based tests to detect and quantify the presence of Anisakis spp. and Pseudoterranova spp. larvae in fish and fish-derived products, including fish fillets, surimi, fish sticks, canned fish, and baby food. Primers and TaqMan MGB probes recognizing only Anisakis spp. and Pseudoterranova spp. were designed on the first internal transcribed spacer 1 regions of rDNA for a real-time polymerase chain reaction assay. A commercial probe for 18S rDNA was used to detect and quantify the total eukaryotic DNA of the samples. The specificity and sensitivity of the assays were tested using reference samples prepared from mixtures made of Anisakis larvae in different quantity of codfish, and subsequent dilutions. Studies were performed to assess the ability of the test to detect and quantify anisakids in various products. Results showed that this test is able to detect anisakid DNA contained in a proportion of 1:10(5) in 1 ng of total DNA. The high prevalence of anisakids reported in main fishery species was confirmed by frequently detecting anisakids DNA in fish muscle and fish-derived products. A partial correlation was found between the number of larvae present in the viscera and the level of contamination of fish fillets. In conclusion, this molecular test is useful to detect the presence of Anisakis spp. and Pseudoterranova spp. in fish and fish-derived products and to quantify the level of contamination along the food chain, with potential applications for fish farms, fish markets, and food producers.

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.

Wolbachia pipientis is known to infect only arthropods and nematodes (mainly filarial worms). A unique feature shared by the two Phyla is the ability to replace the exoskeleton, a process known as ecdysis. This shared characteristic is thought to reflect a common ancestry. Arthropod moulting is induced by the steroid hormone 20-hydroxyecdysone (20E) and a role for ecdysteroids in nematode ecdysis has also been suggested. Removing Wolbachia from filarial worms impairs the host's development. From analyses of the genome of Wolbachia harbored by the filarial nematode Brugia malayi and that of its host, the bacterium may provide a source of heme, an essential component of cytochrome P450's that are necessary for steroid hormone biosynthetic pathways.In arthropods, Wolbachia is a reproductive manipulator, inducing various phenotypic effects that may be due to differences in host physiology, in particular, endocrine-related processes governing development and reproduction. Insect steroids have well-defined roles in the coordination of multiple developmental processes, and in adults they control important aspects of reproduction, including ovarian development, oogenesis, sexual behavior, and in some taxa vitellogenin biosynthesis.According to some authors ecdysteroids may also act as sex hormones. In insects sex differentiation is generally thought to be a strictly genetic process, in which each cell decides its own sexual fate based on its sex chromosome constitution, but, surprisingly, recent data demonstrate that in Drosophila sex determination is not cell-autonomous, as it happens in mammals. Thus the presence of signals coordinating the development of a gender-specific phenotype cannot be excluded.This could explain why Wolbachia interferes with insect reproduction; and also could explain why Wolbachia interferes with insect development.Thus, is "sex (=reproduction) and stripping (=ecdysis)" the key to the intimate relationship between Wolbachia and its host?

The fine structure of spirochete attachments to the plasma membrane of anaerobic protists displays variations here interpreted as legacies of an evolutionary sequence analogous to that from free-living spirochetes to undulipodia (eukaryotic "flagella" and homologous structures). Attached spirochetes form a vestment, a wriggling fringe of motile cells at the edge of the plasma membrane of unidentified cellulolytic protist cells in the hypertrophied hindgut of the digestive system of Mastotermes darwiniensis, the large wood-feeding termite from northern Australia. From the membrane extend both undulipodia and a complex of comparably sized (10-12 microm x 0.2-0.3 microm) ectosymbiotic spirochetes that resembles unruly ciliated epithelium. In the intestines are helical (swimming) and round-body morphotypes. Round bodies (RBs) are slow or immotile spirochetes, propagules known to revert to typical swimming helices under culture conditions favorable for growth. The surfaces of both the spirochete gram-negative eubacteria and the parabasalid protists display distinctive attachment structures. The attached hypertrophied structures, some of which resemble ciliate kinetids, are found consistently at sites where the spirochete termini contact the protist plasma membranes.

Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross-colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body invasion mechanisms independent from host-specific biological characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.

Candidatus Midichloria mitochondrii (M. mitochondrii) belongs to a novel clade of bacteria within the order Rickettsiales. Recent PCR-based screening studies indicate that it is present in a number of blood-sucking arthropods, as well as the blood of some vertebrates. Its medical and veterinary significance remains to be determined. Electron microscopic examinations of M. mitochondrii have thus far been conducted on two infected tick species. Remarkably, the bacterium was found in abundance within the mitochondria of the ovarian cells of each tick species. This makes it the only characterized bacterium able to invade the mitochondria of any multicellular organism. To examine whether mitochondrial invasion is a consistent characteristic of M. mitochondrii, we examined two tick species found in Eastern Australia. One of these species, Ixodes holocyclus, was infected with two M. mitochondrii strains; however, no bacteria were seen in the mitochondria. Comparative studies involving these strains may shed light on the unique phenomenon of mitochondrial invasion.

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.

Background: We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms., Results: DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species., Conclusion: An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

The effects of 5-week doxycycline treatment on the depletion of Wolbachia endobacteria from Onchocerca volvulus, on the interruption of embryogenesis and on microfilariae production, and with regard to macrofilaricidal activity were studied. In 2003, in an endemic area in Ghana, 22 onchocerciasis patients received 100 mg/day doxycycline for 5 weeks. Two years after the start of the study, 20 treated and ten untreated patients were nodulectomized and skin microfilariae were counted. The onchocercomas were examined by immunohistology for the presence of Wolbachia, embryogenesis, and vitality of adult filariae. The latter two parameters were further assessed by alternating logistic regression analysis, taking into account the dependency of worms and nodules in patients. Doxycycline resulted in depletion of Wolbachia and in complete interruption of embryogenesis in all worms that were assumed to have been present during treatment. In the treated patients, only 51% of the female worms were alive, compared to 84% in the untreated patients, indicating a moderate but distinct macrofilaricidal activity of doxycycline at this dose. It is concluded that, in areas with ongoing transmission, doxycycline cannot replace regular ivermectin mass treatment because new infections would require repeated rounds of doxycycline. However, doxycycline can be used for the treatment of individuals outside transmission areas, in foci where ivermectin resistance may occur, and in countries where onchocerciasis and loiasis are co-endemic.

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.

"Candidatus Midichloria mitochondrii," an intracellular symbiont of the tick Ixodes ricinus, is the only described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR to examine cycles of bacterial growth and death throughout the host's development and found that they correspond with the phases of engorgement and molt, respectively.

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.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Symbiotic bacteria have been proposed as tools for control of insect-borne diseases. Primary requirements for such symbionts are dominance, prevalence and stability within the insect body. Most of the bacterial symbionts described to date in Anopheles mosquitoes, the vector of malaria in humans, have lacked these features. We describe an alpha-Proteobacterium of the genus Asaia, which stably associates with several Anopheles species and dominates within the body of An. stephensi. Asaia exhibits all the required ecological characteristics making it the best candidate, available to date, for the development ofa paratransgenic approach for manipulation of mosquito vector competence. Key features of Asaia are: (i) dominance within the mosquito-associated microflora, as shown by clone prevalence in 16S rRNA gene libraries and quantitative real time Polymerase Chain Reaction (qRT-PCR); (ii) cultivability in cell-free media; (iii) ease of transformation with foreign DNA and iv) wide distribution in the larvae and adult mosquito body, as revealed by transmission electron microscopy, and in situ-hybridization experiments. Using a green fluorescent protein (GFP)-tagged Asaia strain, it has been possible to show that it effectively colonizes all mosquito body organs necessary for malaria parasite development and transmission, including female gut and salivary glands. Asaia was also found to massively colonize the larval gut and the male reproductive system of adult mosquitoes. Moreover, mating experiments showed an additional key feature necessary for symbiotic control, the high transmission potential of the symbiont to progeny by multiple mechanisms. Asaia is capable of horizontal infection through an oral route during feeding both in preadult and adult stages and through a venereal pattern during mating in adults. Furthermore, Asaia is vertically transmitted from mother to progeny indicating that it could quickly spread in natural mosquito populations.

Here, we show that an alpha-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Wolbachia pipientis is a maternally inherited, intracellular bacterium found in more than 20 % of all insects, as well as numerous other arthropods and filarial nematodes. It has been the subject of a growing number of studies in recent decades, because of the remarkable effects it has on its arthropod hosts, its potential as a tool for biological control of arthropods of agricultural and medical importance and its use as a target for treatment of filariasis. W. pipientis was originally discovered in cells of the mosquito Culex pipiens and is the only formally described member of the genus. Molecular sequence-based studies have revealed a number of phylogenetically diverse strains of W. pipientis. Owing to uncertainty about whether W. pipientis comprises more than one species, researchers in the field now commonly refer to W. pipientis simply as Wolbachia. In this note, we briefly review higher-level phylogenetic and recombination studies of W. pipientis and propose that all the intracellular symbionts known to cluster closely with the type strain of W. pipientis, including those in the currently recognized supergroups (A-H), are officially given this name.

Sequences of the variable intergenic spacer region 5S (rrfA) 23S (rrlB) rRNA were used to identify Borrelia genospecies present in Ixodes ricinus nymphs collected from the Lamar Lakes area of the Province of Trento, Italy (overall prevalence=6.3%). Four genospecies were identified, one for the first time in this Province (B. valaisiana), and three which have been noted previously (B. afzelii, B. garinii, and B. burgdorferi s.s.). In order to compare the genetic variability of these genospecies in Trento with that at a European level, our 21 sequences (15 new haplotypes) and all appropriate European Borrelia sequences registered in GenBank (up to the end of 2004) were subjected to a phylogenetic analysis (for a total of 73 sequences and 43 haplotypes). Clusters of sequences representing the five main European genospecies (afzelii, garinii, burgdorferi s.s., valaisiana, lusitaniae) are well-supported. At least two other groups of haplotypes (genospecies) are suggested by our analysis; moreover, divergent evolution may be occurring in several genospecies. The maximum uncorrected pairwise differences between sequences within genospecies ranges from 1.5% (B. burgdorferi s.s.), to 2.3% (B. garinii and B. valaisiana) to 4.7% (B. afzelii), and are not correlated with geographical distribution. Within the Province of Trento, these values for the same genospecies are 1.5%, 2.3%, 0.9%, 1.9%, respectively. These high mutation rates within genospecies suggest that the sequencing of haplotypes should continue if we are to fully understand and monitor the evolution and epidemiology of Borrelia.

Polymorphonuclear cells (PMNs) are essential for the innate immune response against invading bacteria. At the same time, modulation of PMNs' apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Wolbachia bacteria are Gram-negative endosymbionts of filarial nematodes and arthropods, phylogenetically related to the genera Anaplasma, Ehrlichia and Neorickettsia (family Anaplasmataceae). Although several pathogens are known to interfere with apoptosis, there is only limited information on specific proteins that modulate this phenomenon. This is the first evidence for the anti-apoptotic activity of a surface protein of Wolbachia from filarial nematode parasites (the Wolbachia surface protein, WSP). The inhibition of apoptosis was demonstrated on purified human PMNs in vitro by different methods. TUNEL assay showed that the percentage of dead cells was reduced after stimulation with WSP; Annexin V-FITC binding assay confirmed that cell death was due mainly to apoptosis and not to necrosis. Reduced caspase-3 activity in stimulated cells also confirmed an inhibition of the apoptotic process.

An intracellular bacterium with the unique ability to enter mitochondria exists in the European vector of Lyme disease, the hard tick Ixodes ricinus. Previous phylogenetic analyses based on 16S rRNA gene sequences suggested that the bacterium formed a divergent lineage within the Rickettsiales (Alphaproteobacteria). Here, we present additional phylogenetic evidence, based on the gyrB gene sequence, that confirms the phylogenetic position of the bacterium. Based on these data, as well as electron microscopy (EM), in situ hybridization and other observations, we propose the name 'Candidatus Midichloria mitochondrii' for this bacterium. The symbiont appears to be ubiquitous in females of I. ricinus across the tick's distribution, while lower prevalence is observed in males (44%). Based on EM and in situ hybridization studies, the presence of 'Candidatus M. mitochondrii' in females appears to be restricted to ovarian cells. The bacterium was found to be localized both in the cytoplasm and in the intermembrane space of the mitochondria of ovarian cells. 'Candidatus M. mitochondrii' is the first bacterium to be identified that resides within animal mitochondria.

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

Woodroaches of the genus Cryptocercus are subsocial and xylophagous cockroaches, distributed in North America and Asia. Studies on male chromosome number in Nearctic species have shown that diploid numbers vary from 2n=37 to 2n=47; numbers from Palearctic species were heretofore unknown. Two hypotheses have been proposed to explain the varying number of chromosomes among Nearctic species: the serial reduction hypothesis, and the parallel scenario. We performed phylogenetic analyses of the COII gene in these species and found evidence for the topology (47(45(43(39,37), which is congruent with the serial reduction hypothesis. We also determined chromosome numbers for the first time in Palearctic species, and found Cryptocercus primarius and Cryptocercus relictus to have relatively low chromosome numbers (2n=17-21) compared to their Nearctic relatives. Finally, our study determined the phylogenetic position of Cryptocercus primarius among other Asian taxa.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Current phylogenies of the intracellular bacteria belonging to the genus Wolbachia identify six major clades (A-F), termed 'supergroups', but the branching order of these supergroups remains unresolved. Supergroups A, B and E include most of the wolbachiae found thus far in arthropods, while supergroups C and D include most of those found in filarial nematodes. Members of supergroup F have been found in arthropods (i.e. termites), and have previously been detected in the nematode Mansonella ozzardi, a causative agent of human filariasis. To resolve the phylogenetic positions of Wolbachia from Mansonella spp., and other novel strains from the flea Ctenocephalides felis and the filarial nematode Dipetalonema gracile, the authors generated new DNA sequences of the Wolbachia genes encoding citrate synthase (gltA), heat-shock protein 60 (groEL), and the cell division protein ftsZ. Phylogenetic analysis confirmed the designation of Wolbachia from Mansonella spp. as a member of the F supergroup. In addition, it was found that divergent lineages from Dip. gracile and Cte. felis lack any clear affiliation with known supergroups, indicating further genetic diversity within the Wolbachia genus. Finally, although the data generated did not permit clear resolution of the root of the global Wolbachia tree, the results suggest that the transfer of Wolbachia spp. from arthropods to nematodes (or vice versa) probably occurred more than once.

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.

Filarial nematodes are important helminth parasites of the tropics and a leading cause of global disability. They include species responsible for onchocerciasis, lymphatic filariasis and dirofilariasis. A unique feature of these nematodes is their dependency upon a symbiotic intracellular bacterium, Wolbachia, which is essential for normal development and fertility. Advances in our understanding of the symbiosis of Wolbachia bacteria with filarial nematodes have made rapid progress in recent years. Here we summarise our current understanding of the evolution of the symbiotic association together with insights into the functional basis of the interaction derived from genomic analysis. Also we discuss the contribution of Wolbachia to inflammatory-mediated pathogenesis and adverse reactions to anti-filarial drugs and describe the outcome of recent field trials using antibiotics as a promising new tool for the treatment of filarial infection and disease.

A 12-year-old, 13 kg, mixed-breed male dog was referred for anorexia and depression. The dog showed discomfort on abdominal palpation. Abdominal ultrasound examination revealed multiple, small, round anechoic cystic structures. Cystic fluid obtained with fine needle aspiration contained several 2-4 mm white motile flecks. Microscopic examination of the fluid revealed numerous irregularly shaped organisms measuring several hundred microns to 3 mm, the morphology of which was suggestive of intact and fragmented acephalic metacestodes of the genus Mesocestoides sp. Molecular analysis confirmed that the peritoneal infection was caused by Mesocestoides sp.

This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology(WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification,diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.

Wolbachia are obligatory endosymbionts in many species of filarial nematodes. Certain bacterial molecules induce antibody responses in mammalian hosts infected with filariae, while others activate inflammatory responses that contribute to pathology. These findings, coupled with antibiotic studies demonstrating the dependence of filarial embryogenesis on the presence of Wolbachia, have intensified research on Wolbachia-nematode interactions, and the effects of Wolbachia molecules on the mammalian immune system. By amplification and sequencing of 16S rDNA and catalase sequences, we show that filarial DNA samples prepared from nematodes collected under typical conditions are frequently contaminated with Pseudomonas DNA. Analysis of a published DNA fragment containing a catalase attributed to the Wolbachia of Onchocerca volvulus showed it to be most like Pseudomonas, both in terms of sequence similarity and genomic organization. Additionally, there was no obvious catalase in either of two available Wolbachia genome sequences. Contamination of filarial DNA with bacterial sequences other than Wolbachia can complicate studies of the role of these symbionts in filarial biology.

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).

More than 150 million humans in tropical countries are infected by filarial nematodes which harbor intracellular bacterial endosymbionts of the genus Wolbachia (Rickettsiales). These bacteria have been implicated in adverse effects of drug treatment in filariasis. The present study provides evidence that purified major Wolbachia surface protein (rWSP) acts as an inducer of the innate immune system through TLR2 and TLR4: 1) recombinant, stringently purified rWSP elicited the release of TNF-alpha, IL-12, and IL-8 from cultured blood cells of both Onchocerca volvulus-infected and uninfected people; 2) the inflammatory response to rWSP challenge was TLR2- and TLR4-dependent as demonstrated with TLR-transfected fibroblastoid cells, as well as macrophages and dendritic cells from functional TLR-deficient mice; 3) blood cells of onchocerciasis patients exposed to rWSP also generated down-regulating mediators IL-10 and PGE(2) after 6 days of culture; 4) furthermore, rWSP-reactive IgG1 Abs were present in sera of O. volvulus-infected people but not in those of uninfected Europeans. The lack of rWSP-reactive IgE and IgG4 in serum indicated a bias toward a Th1-type adaptive immune response. Abs against rWSP stained endobacteria in living and degenerating adult O. volvulus filariae, tissue microfilariae and host tissue macrophages that apparently had engulfed microfilariae. Thus, filarial helminths, through products of their endobacteria such as WSP, acquire characteristics of a typical microbial pathogen inducing immune responses via TLR2 and TLR4.

Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

Sexual transmission occurs commonly in microparasites such as viruses and bacteria, but this is an unusual transmission route for macroparasites. Here we present evidence which suggests that a nematode parasite of Wood Mice (Apodemus sylvaticus) may be sexually transmitted and we have classified the nematode using molecular data. Wood Mice were collected annually in the course of work on their reproductive physiology. Larval nematodes were found in the epididymides of 19.6% of males. It seems likely that they would be transmitted to females at ejaculation. To identify these larval nematodes, which we were unable to do using morphological features, we sequenced the 18S rDNA. Sequence comparisons with the molecular phylogeny of Blaxter et al. (1998) demonstrated that they were bursate nematodes (Order Strongylida). The relationships between strongylid taxa were poorly resolved by 18S rDNA. However, both distance and parsimony analyses grouped the nematode with the superfamily Metastrongylidea in a clade containing Filaroides and Angiostrongylus sp. Importantly, the sequences were distinct from those of Heligmosomoides polygyrus and Angiostrongylus dujardini, two common strongylid nematodes of Apodemus. We were therefore unable positively to identify these worms by matching their sequences with those from morphologically identifiable adult strongylid nematodes infecting Apodemus. These results demonstrate that an as yet unidentified strongylid is quite commonly found in large numbers in the male reproductive tract of Wood Mice. Further work is required to understand the biology and transmission dynamics of this interesting system.