Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

The mycobiota of the sand fly Phlebotomus perniciosus: Involvement of yeast symbionts in uric acid metabolism.

The knowledge of the fungal mycobiota of arthropods, including the vectors of human and animal diseases, is still limited. Here, the mycobiota associated with the sand fly Phlebotomus perniciosus, the main vector of leishmaniasis in the western Mediterranean area, by a culture-dependent approach (microbiological analyses and sequencing of the 26S rRNA gene), internal transcribed spacer (ITS) rRNA amplicon-based next-generation sequencing, fluorescence in situ hybridisation (FISH), and genome sequencing of the dominant yeast species was investigated. The dominant species was Meyerozyma guilliermondii, known for its biotechnological applications. The focus was on this yeast and its prevalence in adults, pupae and larvae of reared sand flies (overall prevalence: 57.5%) and of field-collected individuals (overall prevalence: 9%) was investigated. Using whole-mount FISH and microscopic examination, it was further showed that M. guilliermondii colonizes the midgut of females, males and larvae and the distal part of Malpighian tubules of female sand flies, suggesting a possible role in urate degradation. Finally, the sequencing and analysis of the genome of M. guilliermondii allowed predicting the complete uric acid degradation pathway, suggesting that the yeast could contribute to the removal of the excess of nitrogenous wastes after the blood meal of the insect host.

NMR as evaluation strategy for cellular uptake of nanoparticles.

Advanced nanostructured materials, such as gold nanoparticles, magnetic nanoparticles, and multifunctional materials, are nowadays used in many state-of-the-art biomedical application. However, although the engineering in this field is very advanced, there remain some fundamental problems involving the interaction mechanisms between nanostructures and cells or tissues. Here we show the potential of (1)H NMR in the investigation of the uptake of two different kinds of nanostructures, that is, maghemite and gold nanoparticles, and of a chemotherapy drug (Temozolomide) in glioblastoma tumor cells. The proposed experimental protocol provides a new way to investigate the general problem of cellular uptake for a variety of biocompatible nanostructures and drugs.

"Candidatus Midichloriaceae" fam. nov. (Rickettsiales), an ecologically widespread clade of intracellular alphaproteobacteria.

"Candidatus Midichloria mitochondrii" is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus. Bacteria phylogenetically related to "Ca. Midichloria mitochondrii" (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales. This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family "Candidatus Midichloriaceae." In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Gill-associated bacteria are homogeneously selected in amphibious mangrove crabs to sustain host intertidal adaptation.

BACKGROUND: The transition from water to air is a key event in the evolution of many marine organisms to access new food sources, escape water hypoxia, and exploit the higher and temperature-independent oxygen concentration of air. Despite the importance of microorganisms in host adaptation, their contribution to overcoming the challenges posed by the lifestyle changes from water to land is not well understood. To address this, we examined how microbial association with a key multifunctional organ, the gill, is involved in the intertidal adaptation of fiddler crabs, a dual-breathing organism. RESULTS: Electron microscopy revealed a rod-shaped bacterial layer tightly connected to the gill lamellae of the five crab species sampled across a latitudinal gradient from the central Red Sea to the southern Indian Ocean. The gill bacterial community diversity assessed with 16S rRNA gene amplicon sequencing was consistently low across crab species, and the same actinobacterial group, namely Ilumatobacter, was dominant regardless of the geographic location of the host. Using metagenomics and metatranscriptomics, we detected that these members of actinobacteria are potentially able to convert ammonia to amino acids and may help eliminate toxic sulphur compounds and carbon monoxide to which crabs are constantly exposed. CONCLUSIONS: These results indicate that bacteria selected on gills can play a role in the adaptation of animals in dynamic intertidal ecosystems. Hence, this relationship is likely to be important in the ecological and evolutionary processes of the transition from water to air and deserves further attention, including the ontogenetic onset of this association. Video Abstract.

Phylogenomic evidence for the presence of a flagellum and cbb(3) oxidase in the free-living mitochondrial ancestor.

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

A study on the presence of flagella in the order Rickettsiales: the case of 'Candidatus Midichloria mitochondrii'.

According to Bergey's Manual of Systematic Bacteriology, the Rickettsiales are '…bacteria with typical gram-negative cell walls and no flagella'. The recently sequenced genome of 'Candidatus Midichloria mitochondrii', a divergent lineage within the order Rickettsiales capable of invading mitochondria in ixodid ticks, revealed the presence of 26 putative flagellar genes. Open questions in relation to this observation are whether these genes are expressed and whether they possess the domains expected for the flagellar function. Here we show that: (a) the putative flagellar proteins of 'Ca. M. mitochondrii' actually possess the conserved domains and structural features required for their function in a model bacterium; (b) the seven flagellar genes of 'Ca. M. mitochondrii' that have been tested are expressed at the RNA level; and (c) the putative flagellar cap gene of this bacterium (FliD) is expressed at the protein level, and can be stained within the bacterium and at its surface. Beside the specific questions that we have addressed that relate to the first evidence, to our knowledge, for a flagellar apparatus in a member of the order Rickettsiales, we present here novel tools (recombinant protein and antibodies) that will facilitate the study of 'Ca. M. mitochondrii'.

Morphological characteristics and abundance of prokaryotes associated with gills in mangrove brachyuran crabs living along a tidal gradient.

Due to the chemico-physical differences between air and water, the transition from aquatic life to the land poses several challenges for animal evolution, necessitating morphological, physiological and behavioural adaptations. Microbial symbiosis is known to have played an important role in eukaryote evolution, favouring host adaptation under changing environmental conditions. We selected mangrove brachyuran crabs as a model group to investigate the prokaryotes associated with the gill of crabs dwelling at different tidal levels (subtidal, intertidal and supratidal). In these animals, the gill undergoes a high selective pressure, finely regulating multiple physiological functions during both animal submersion under and emersion from the periodical tidal events. We hypothesize that similarly to other marine animals, the gills of tidal crabs are consistently colonized by prokaryotes that may quantitatively change along the environmental gradient driven by the tides. Using electron microscopy techniques, we found a thick layer of prokaryotes over the gill surfaces of all of 12 crab species from the mangrove forests of Saudi Arabia, Kenya and South Africa. We consistently observed two distinct morphotypes (rod- and spherical-shaped), positioned horizontally and/or perpendicularly to the gill surface. The presence of replicating cells indicated that the prokaryote layer is actively growing on the gill surface. Quantitative analysis of scanning electron microscopy images and the quantification of the bacterial 16S rRNA gene by qPCR revealed a higher specific abundance of prokaryote cells per gill surface area in the subtidal species than those living in the supratidal zone. Our results revealed a correlation between prokaryote colonization of the gill surfaces and the host lifestyle. This finding indicates a possible role of prokaryote partnership within the crab gills, with potential effects on animal adaptation to different levels of the intertidal gradient present in the mangrove ecosystem.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi.

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois noir in Vitis vinifera.

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.

Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control.

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.

Detection of a morphogenetically novel Sarcocystis hominis-like in the context of a prevalence study in semi-intensively bred cattle in Italy.

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

Modeling the Life Cycle of the Intramitochondrial Bacterium "Candidatus Midichloria mitochondrii" Using Electron Microscopy Data.

"Candidatus Midichloria mitochondrii" is a Gram-negative bacterium that lives in strict intracellular symbiosis with the hard tick Ixodes ricinus, forming one of the most intriguing endosymbiosis described to date. The bacterium is capable of durably colonizing the host mitochondria, a peculiar tropism that makes "Ca. Midichloria mitochondrii" a very interesting tool to study the physiology of these cellular organelles. The interaction between the symbiont and the organelle has, however, been difficult to characterize. A parallelism with the predatory bacterium Bdellovibrio bacteriovorus has been drawn, suggesting the hypothesis that "Ca. Midichloria mitochondrii" could prey on mitochondria and consume them to multiply. We studied the life cycle of the bacterium within the host oocytes using a multidisciplinary approach, including electron microscopy, molecular biology, statistics, and systems biology. Our results were not coherent with a predatory-like behavior by "Ca. Midichloria mitochondrii" leading us to propose a novel hypothesis for its life cycle. Based on our results, we here present a novel model called the "mitochondrion-to-mitochondrion hypothesis." Under this model, the bacterium would be able to move from mitochondrion to mitochondrion, possibly within a mitochondrial network. We show that this model presents a good fit with quantitative electron microscopy data. IMPORTANCE Our results suggest that "Candidatus Midichloria mitochondrii," the intramitochondrial bacterium, does not invade mitochondria like predatory bacteria do but instead moves from mitochondrion to mitochondrion within the oocytes of Ixodes ricinus. A better understanding of the lifestyle of "Ca. Midichloria mitochondrii" will allow us to better define the role of this bacterial symbiont in the host physiology.

Identification of a Novel Brevibacillus laterosporus Strain With Insecticidal Activity Against Aedes albopictus Larvae.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.

Acetic acid bacteria, newly emerging symbionts of insects.

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.

Mosquito-bacteria symbiosis: the case of Anopheles gambiae and Asaia.

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.

Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders.

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.

Acetobacter tropicalis is a major symbiont of the olive fruit fly (Bactrocera oleae).

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.

Chlamydia infection and lymphomas: association beyond ocular adnexal lymphomas highlighted by multiple detection methods.

PURPOSE: Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamydia detection tools to identify Cp elementary bodies-containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. EXPERIMENTAL DESIGN: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. RESULTS: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact Cp elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyer's ring. CONCLUSIONS: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated.

Bacteria of the genus Asaia: a potential paratransgenic weapon against malaria.

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.