Search

Keywords: Midichloriaceae family, Oncorhynchusmykiss, real-time PCR, red mark syndrome.Red mark syndrome (RMS) is a chronic skin dis-ease of unknown aetiology affecting farmed rain-bow trout Oncorhynchus mykiss (Walbaum) inEurope. It consists of single or multiple skinlesions usually localized on the trunk of fishapproaching market size. RMS was first reportedin Scotland in 2003 but has subsequently beenobserved in other European countries, includingItaly (Verner-Jeffreys et al. 2008; Schmidt-Post-haus et al. 2009). This disease is generally associ-ated with water temperatures below 16 °C andcan still be present at 2 °C (Verner-Jeffreys et al.2008; McCarthy et al. 2013). RMS causes brightred lesions that tend to heal spontaneously, withno effects on weight or behaviour and the healingtime seems to be associated with the water tem-perature (Ferguson et al. 2006). Histologically,severe lymphohistiocytic dermatitis is reportedwith scale resorption (Ferguson et al. 2006; Nogu-era 2008; Verner-Jeffreys et al. 2008; Schmidt-Posthaus et al. 2010). In some RMS-affected indi-viduals, acute necrotizing myocarditis,inflammation of intestinal muscle, splenic conges-tion, peritonitis and perivascular lymphocyte infil-trate can be observed (Oidtmann et al. 2013).Although RMS is not lethal, its morbidity canreach up to 60% in fish culture and can thuscause important economic losses, due to down-grading of the product (Schmidt-Posthaus et al.2009; Oidtmann et al. 2013). In the UnitedStates, a similar condition of O. mykiss is knownas strawberry disease (SD) and the skin pathologyassociated with it resembles that of RMS.No aetiological agent has been unequivocallyidentified for RMS or for SD, although differentauthors suggest that a single transmissible agentcould be responsible for both (Lloyd et al. 2011).Oman (1990) reported experimental infectionthrough inoculation with SD lesion homogenate(Lloyd et al. 2008). Ferguson et al. (2006) sug-gested a possible association with Flavobacteriumpsychrophilum, but no data confirmed this hypoth-esis. Experimental cohabitation between fishaffected by RMS and na€ive ones demonstrated thetransmissibility of this pathology (Verner-Jeffreyset al. 2008). Moreover, histological analysisshowed acute inflammation in the area of the skinlesion with the presence of neutrophils and pro-tein oedema (Metselaar et al. 2010), suggesting abacterial infection.Lloyd et al. (2008) presented PCR evidence forthe presence of a Rickettsia-like organism (RLO)

a b s t r a c t Background: Liver fibrosis is accelerated in patients co-infected with human immunodeficiency virus and hepatitis C viruses. Aims: We investigated the correlation between liver fibrosis, immune activation and microbial translo- cation. Methods: This cross-sectional study included patients with hepatitis C virus (HCV) and human immuno- deficiency virus (HIV) mono-infections, HIV/HCV co-infection, and healthy controls (20 subjects/group). Peripheral blood was analysed to determine the levels of Forkhead box 3 (Foxp3) T cells, TGF-1, CD14 (soluble and surface isoforms), IL-17 and bacterial translocation products. These measurements were correlated to the severity of liver fibrosis, measured with the FIB-4 score and transient elastography. Results: Foxp3T cell levels were significantly elevated in HIV mono-infected and co-infected groups (p < 0.0005). FIB-4 and liver stiffness values inversely correlated with TGF-1 (p = 0.0155 and p = 0.0498). Bacterial DNA differed significantly in the HIV-positive compared to the other groups: HIV/HCV co- infected subjects had significantly higher serum levels of bacterial translocation products, CD14, and IL-17 levels (p < 0.001). Conclusions: Fibrosis stage in HIV/HCV co-infection may be influenced by immune activation due either by viral infections or to bacterial translocation.

In a previous study, dogs experimentally infected with Dirofilaria immitis were treated with either ivermectin or doxycycline or a combination of both. The adulticide effect was significantly higher in the dogs treated with both drugs and was similar to that observed in dogs treated with melarsomine hydrochloride. In the present study, lung tissue samples from these dogs were evaluated for the presence of T regulatory (Foxp3+) cells by immunohistochemistry. Cells were enumerated for each dog in the four groups and compared with untreated controls. There was a significantly lower number of Treg cells in those dogs treated with a combination of both drugs when compared either to the control group or to the other groups treated with either drug alone or with melarsomine. These results suggest that successful adulticide effects of doxycycline and ivermectin are associated with a decrease in immune regulation towards the parasite.

We have amplified by PCR the sequences of the 5 S ribosomal spacer of Setaria labiatopapillosa and Foleyella furcata. After sequencing, these sequences have been compared with those of Dirofilaria immitis and Dirofilaria repens. Two major goals have been achieved: (i) the establishment of a multiplex PCR-based diagnostic assay, applicable to identify the four species in vertebrate and invertebrate hosts; (ii) the identification, in S. labiatopapillosa and F. furcata, of a canonical spliced leader 1 (SL1) sequence, so confirming that only D. repens, of the filarial parasites so far studied, shows a peculiar SL1 sequence. The PCR assay here developed and the analysis of the 5 S ribosomal spacer, can further improve both epidemiological and molecular analysis of these filarial species.

Peripheral blood samples were collected randomly from 195 horses in various parts of Hungary, and the presence of microfilariae was evaluated by the Knott technique. On the basis of morphological identification 18 of the horses (9·2 per cent) were infected with Setaria equina , and the infection was confirmed in 10 animals by pcr and sequencing. The level of microfilaraemia was between 1 and 1138 larvae in 2 ml of blood. There was no correlation between the time of sampling or the sex of the animals (stallions versus mares) and the prevalence of infection, but the prevalence decreased with age. There was a significant association between the prevalence of microfilaraemia and the presence of still waters; positive samples were collected either in the region of Lake Balaton, the largest lake in the country, or at places with nearby ponds.

Oreina cacaliae (Coleoptera: Chrysomelidae) is a rare example of viviparous insect, able to feed on toxic plants and sequester toxic compounds. Here we present the results of a study on the microbiota associated with O. cacaliae, based on 16S rRNA bacterial gene sequencing. Wolbachia resulted as the dominant bacterium, both in males (100%) and in females (91.9%). Based on multilocus sequence typing, the detected Wolbachia was described as a new sequence type (Wolbachia Ocac_A_wVdO). Phylogenetic analyses assigned Wolbachia Ocac_A_wVdO to supergroup-A. In situ hybridization and electron microscopy confirmed the presence of Wolbachia within O. cacaliae oocytes, indicating its transovarial transmission. PCR specific for Wolbachia was performed on representatives of six species of Oreina; the presence / absence of Wolbachia was then mapped on a cladogram representing the phylogeny of the insect host. Finally, since viviparous species of Oreina were either infected or non-infected by Wolbachia, we cannot derive any conclusion about the possibility that this symbiont played some role in the evolution of viviparity.

Background The Midichloriaceae is a novel family of the order Rickettsiales, that encompasses intracellular bacteria associated with hard ticks (Ixodidae) and other arthropods. The most intensively investigated member of this family is Midichloria mitochondrii, a symbiotic bacterium of the sheep tick Ixodes ricinus, characterized by the capacity of multiplying inside the mitochondria. A recent study suggested that these bacteria might be inoculated into the human host during the tick bite. The purpose of this study was to determine the potential infectivity of Midichloria bacteria for non-human animals exposed to the risk of tick bite. Methods Blood from horses, cattle, sheep and dogs exposed to the risk of tick bite was included in this study. DNAs were extracted, and amplified using 16S ribosomal RNA primers conserved in the Midichloria genus. Furthermore, sera from dogs exposed to the risk of tick bite were analyzed in order to evaluate the presence of antibodies against the recombinant flagellar protein (rFliD) from M. mitochondrii using an ELISA test. Results Here we present two lines of evidence that support the possibility that bacteria from the genus Midichloria are inoculated into vertebrate hosts during a tick bite: (i) a direct evidence, i.e. the detection of circulating DNA from bacteria related with M. mitochondrii, in the blood of vertebrates exposed to tick parasitism; (ii) a further indirect evidence, i.e. the presence of antibodies against an antigen from M. mitochondrii in dogs exposed to the risk of tick bite. It is interesting to note that variability was detected in the Midichloria gene sequences recovered from positive animals, and that some of these sequences were identical to those generated from tick-associated Midichloria. Conclusions Based on the results, and on the overall information so far published on the genus Midichloria, we suggest that these bacteria are likely to represent a novel group of vector-borne agents, with the potential of infecting mammalian hosts. Whether inoculation of Midichloria bacteria could cause a true infection and pathological alteration in mammalian hosts is still to be determined. Surely, results emphasize the relevance of Midichloria bacteria in investigations on tick immunology and tick-bite markers.

“ 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.

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’.

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 alpha-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.

Comparing the 5S ribosomal RNA spacer of Dirofilaria repens and D. immitis, we found that in D. repens PCR generates two amplification products. In both products, the putative spliced leader 1 sequences (SL1) show some nucleotide substitutions compared to the canonical SL1 sequence of nematodes. SL I is a short sequence transferred to the 5' end of most messenger RNAs through a trans-splicing reaction. All the other filariae studied thus far amplify a single fragment showing a canonical SL1 sequence. D. repens appears thus peculiar.

Genetic variability of the ovine parasiteHaemonchus contortusfrom the Alpine area was investigated using mitochondrial DNA (nd4gene), internal transcribed spacers 1 and 2 and microsatellites, in order to assess whether cross-transmission between domestic and wild ruminants occurs. The dataset was composed of 78 individual adult maleH. contortuscollected from chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) from different alpine areas. The data obtained show low host specificity and high genetic variation withinH. contortuspopulations. The analyses indicate the presence of two mitochondrial haplotype clusters among host species and the absence of cryptic parasite species, confirmingH. contortusas a generalist nematode and suggesting that parasite transmission between populations of domestic and wild ruminants normally occurs.

Zeta-chain-associated protein (ZAP-70) and spleen tyrosine kinase (Syk) are structurally and functionally homologous tyrosine kinases playing a role in the T- and B-cell signal transduction. Their activation can lead to lymphokine production, cytolitic activity, antibody secretion, cell proliferation, differentiation, survival and phagocytosis. Anomalous ZAP-70 and Syk expression is reported to be related to tumor formation and progression, and ZAP-70 immunoreactivity is a good prognostic marker of disease progression in human chronic lymphocytic leukaemia (CLL). Until now, to our knowledge, there are no reports about canine ZAP-70 and Syk expression profiles. In the present study, a RT-PCR procedure for the quali-quantitative evaluation of canine ZAP-70 and Syk transcripts was designed. The expression patterns of canine ZAP-70 and Syk mRNAs were evaluated in canine leukocyte subpopulations and in peripheral whole blood samples from healthy dogs and from dogs with different types of leukaemia. Similarly to humans, normal canine CD4+ and CD8+ T cells showed high expression of ZAP-70, whereas Syk was abundantly expressed in normal CD21+ B cells. The expression profile of ZAP-70 and Syk was markedly different in canine normal and leukaemic blood. Decreased Syk expression was detected in dogs with T-cell CLL, whereas decreased ZAP-70 expression was detected in dogs with B-cell CLL and B-cell acute lymphocytic leukaemia (ALL). The comparison of ZAP-70 and Syk mRNA levels between normal and leukaemic peripheral whole blood showed that the expression ratio ZAP-70/Syk is subjected to modification depending on the leukaemia status of patients. The results of the present work open an interesting topic for leukaemogenesis investigation and are the basis for further studies for a proper evaluation of the potential utility of these parameters for the diagnosis and prognosis of canine T- and B-cell leukaemia.

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0–6, 10–12, 16–18, 22–26, and 28–34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24 h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.

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.

Accurate diagnosis of canine filariosis is essential for choosing correct therapeutic approach. Therefore, reliable methods for discriminating among the different filarial infections in dogs are needed. The authors report simple and highly specific molecular methods that identify the three most common filarial nematodes of European dogs: Dirofilaria immitis, D. repens and Acanthocheilonema (syn. Dipetalonema) reconditum, based on (1) PCR amplifications of mitochondrial DNA (12S rDNA and coxI) with general filarial primers followed by digestion with restriction enzymes that generates band polymorphisms clearly discriminating the three species and (2) PCR amplifications with species-specific primers to support the restriction analysis, in particular in the case of multiple infections. (c) 2006 Elsevier B.V. All rights reserved.

Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.

The bacterial endosymbiont Wolbachia of several species of filarial nematodes plays an important role in the inflammatory pathology of filariasis. Nitric oxide (NO) production has also been implicated in the immune response during filarial infections. Here we present data indicating that a recombinant Wolbachia surface protein (rWSP) induces iNOs mRNA expression and NO production, as well as IFN-γ and a Th1-type antibody response, in inoculated BALB/c mice. This effect is not observed when mice are inoculated with a recombinant heat shock protein from Wolbachia (GroEL).

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility.

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-α, 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 PGE2 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.

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, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.

BACKGROUND: Many filarial nematodes harbour Wolbachia endobacteria. These endobacteria are transmitted vertically from one generation to the next. In several filarial species that have been studied to date they are obligatory symbionts of their hosts. Elimination of the endobacteria by antibiotics interrupts the embryogenesis and hence the production of microfilariae. The medical implication of this being that the use of doxycycline for the treatment of human onchocerciasis and bancroftian filariasis leads to elimination of the Wolbachia and hence sterilisation of the female worms. Wolbachia play a role in the immunopathology of patients and may contribute to side effects seen after antifilarial chemotherapy. In several studies Wolbachia were not observed in Loa loa. Since these results have been doubted, and because of the medical significance, several independent methods were applied to search for Wolbachia in L. loa. METHODS: Loa loa and Onchocerca volvulus were studied by electron microscopy, histology with silver staining, and immunohistology using antibodies against WSP, Wolbachia aspartate aminotransferase, and heat shock protein 60. The results achieved with L. loa and O. volvulus were compared. Searching for Wolbachia, genes were amplified by PCR coding for the bacterial 16S rDNA, the FTSZ cell division protein, and WSP. RESULTS: No Wolbachia endobacteria were discovered by immunohistology in 13 male and 14 female L. loa worms and in numerous L. loa microfilariae. In contrast, endobacteria were found in large numbers in O. volvulus and 14 other filaria species. No intracellular bacteria were seen in electron micrographs of oocytes and young morulae of L. loa in contrast to O. volvulus. In agreement with these results, Wolbachia DNA was not detected by PCR in three male and six female L. loa worms and in two microfilariae samples of L. loa. CONCLUSIONS: Loa loa do not harbour obligatory symbiotic Wolbachia endobacteria in essential numbers to enable their efficient vertical transmission or to play a role in production of microfilariae. Exclusively, the filariae cause the immunopathology of loiasis is patients and the adverse side effects after antifilarial chemotherapy. Doxycycline cannot be used to cure loiais but it probably does not represent a risk for L. loa patients when administered to patients with co-infections of onchocerciasis.

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

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

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

The dog parasite Dirofilaria immitis can infect humans. Patients with pulmonary dirofilariasis were tested for immunoglobulin G (IgG) antibodies against the surface protein of Wolbachia , the bacterial endosymbiont of D . immitis . These patients showed significantly higher IgG titers than healthy individuals from areas in which D . immitis was endemic as well as areas in which it was not endemic. Titration of anti- Wolbachia surface protein IgG could become useful for diagnostic applications.

Background Wolbachiaendosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. Results Here we show that recombinant Wolbachia Surface Protein (WSP) stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiaecells. Conclusions We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae) while a milder elicitor in a naturally-infected species (Aedes albopictus). Since the WSP of a mosquito non-native (nematode) Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.

Echinococcus granulosus is the aetiological agent of cystic echinococcosis (CE), which is a public health problem in many eastern European countries, particularly in Romania, where the infection causes a high number of human and animal cases. To shed light on the transmission patterns of the parasite, we performed a genotyping analysis on 60 cyst samples obtained from patients who live in south-eastern Romania and who underwent surgery for liver or lung CE. DNA was extracted from the endocysts or the cyst fluids, and fragments of cytochrome c oxidase subunit 1 and NADH dehydrogenase subunit 1 mitochondrial genes ( cox1 and nd1 , respectively) were amplified by PCR and sequenced. We found that most of the samples analysed (59/60) belonged to the G1–G3 complex ( E. granulosus sensu stricto), which contains the most widespread and infective strains of the parasite. We also identified the first human patient infected by a non-G1–G3 genotype of E. granulosus in this country. As the DNA sequence of this cyst sample showed maximum homology with the G6–G10 complex ( Echinococcus canadensis ), this is, in all likelihood, a G7 genotype, which is often found in pigs and dogs in most countries of eastern and south-eastern Europe.

The yeast Wickerhamomyces anomalus has been proposed for many biotechnological applications in the food industry. However, a number of opportunistic pathogenic strains have been reported as causative agents of nosocomial fungemia. Recognition of potentially pathogenic isolates is an important challenge for the future commercialization of this yeast. The isolation of W. anomalus from different matrices and, recently, from mosquitoes, requires further investigations into its circulation in humans. Here we present a qPCR protocol for the detection of W. anomalus in human blood samples and the results of a screening of 525 donors, including different classes of patients and healthy people.

International audience