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BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

An anti-tick mRNA cocktail vaccine promotes tick detachment and prevents transmission of tick-borne infection in guinea pigs (Sajid et al ).

Hematophagous arthropods are responsible for the transmission of a variety of pathogens that cause disease in humans and animals. Ticks of the Ixodes ricinus complex are vectors for some of the most frequently occurring human tick-borne diseases, particularly Lyme borreliosis and tick-borne encephalitis virus (TBEV). The search for vaccines against these diseases is ongoing. Efforts during the last few decades have primarily focused on understanding the biology of the transmitted viruses, bacteria and protozoans, with the goal of identifying targets for intervention. Successful vaccines have been developed against TBEV and Lyme borreliosis, although the latter is no longer available for humans. More recently, the focus of intervention has shifted back to where it was initially being studied which is the vector. State of the art technologies are being used for the identification of potential vaccine candidates for anti-tick vaccines that could be used either in humans or animals. The study of the interrelationship between ticks and the pathogens they transmit, including mechanisms of acquisition, persistence and transmission have come to the fore, as this knowledge may lead to the identification of critical elements of the pathogens' life-cycle that could be targeted by vaccines. Here, we review the status of our current knowledge on the triangular relationships between ticks, the pathogens they carry and the mammalian hosts, as well as methods that are being used to identify anti-tick vaccine candidates that can prevent the transmission of tick-borne pathogens.

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

Lyme borreliosis is an emerging tick-borne disease caused by spirochetes Borrelia burgdorferi sensu lato. In Europe, Lyme borreliosis is predominantly caused by Borrelia afzelii and transmitted by Ixodes ricinus. Although Borrelia behavior throughout tick development is quite well documented, specific molecular interactions between Borrelia and the tick have not been satisfactorily examined. Here, we present the first transcriptomic study focused on the expression of tick midgut genes regulated by Borrelia. By using massive analysis of cDNA ends (MACE), we searched for tick transcripts expressed differentially in the midgut of unfed, 24h-fed, and fully fed I. ricinus nymphs infected with B. afzelii. In total, we identified 553 upregulated and 530 downregulated tick genes and demonstrated that B. afzelii interacts intensively with the tick. Technical and biological validations confirmed the accuracy of the transcriptome. The expression of five validated tick genes was silenced by RNA interference. Silencing of the uncharacterized protein (GXP_Contig_30818) delayed the infection progress and decreased infection prevalence in the target mice tissues. Silencing of other genes did not significantly affect tick feeding nor the transmission of B. afzelii, suggesting a possible role of these genes rather in Borrelia acquisition or persistence in ticks. Identification of genes and proteins exploited by Borrelia during transmission and establishment in a tick could help the development of novel preventive strategies for Lyme borreliosis.

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.

A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.

By searching nucleotide databases for the North American Lyme disease vector, Ixodes scapularis, we have complemented the previously characterized European Ixodes ricinus legumain IrAE1 with a full set of nine analogous genes (isae1-9). Six of these were PCR confirmed as genes present in all tick genomes tested. The absolute mRNA copy number examined by quantitative (q)PCR enabled expression profiling and an absolute comparison of mRNA levels for individual I. scapularis (Is)AEs in tick tissues. Four IsAEs (1, 2, 4, 9) were expressed solely in the gut and thus are proposed to be involved in host blood digestion. Expression qPCR profiling over developmental stages confirmed IsAE1, the direct analogue of previously characterized I. ricinus IrAE1, as the principle legumain transcript in partially engorged females, and demonstrated its strong regulation by on-host feeding in larvae, nymphs and females. In contrast, IsAE2 was the predominant gut legumain in unfed nymphs, unfed females and males. In-silico, IsAE1 and IsAE2 protein three-dimensional structural models displayed minimal differences in overall proenzyme structures, even in comparison with recently resolved crystal structures of mammalian prolegumain. Three functional studies were performed in I. ricinus with IsAE1/IsAE2 analogues: double IrAE1/IrAE2 RNA interference silencing, feeding of ticks on IrAE1+IrAE2 immunized hosts and in vitro membrane tick feeding on blood containing a legumain-specific inhibitor. The latter experiment led to reduced weights of fully engorged ticks and limited oviposition, and indicated the potential of legumain inhibitors for novel anti-tick interventions.

Ticks possess components of a primordial complement system that presumably play a role in the interaction of the tick immune system with tick-borne pathogens and affect their transmission. Here we characterized a novel complement component, tagged as IrC2/Bf, from the hard tick Ixodes ricinus , the principal vector of Lyme disease in Europe. IrC2/Bf is a multi-domain molecule composed of 5–7 CCP modules, varied by alternative splicing, followed by a von Willebrand factor A domain and a C-terminal trypsin-like domain. The primary structure and molecular architecture of IrC2/Bf displays the closest homology to the C3-complement component convertases described in horseshoe crabs. The irc2/bf gene is mainly expressed in the tick fat body associated with the trachea and, as determined by western blotting, the protein is present in low amounts in tick hemolymph. Expression of irc2/bf mRNA was significantly up-regulated in response to the intra-hemocoelic injection of the yeast Candida albicans and all tested Borrelia sp. strains ( B. burgdorferi NE5264, B. burgdorferi CB26, B. garinii MSLB, B. afzelii CB43), but was not affected by injection of model Gram-negative and Gram-positive bacteria or the aseptic injection control. In-line with these results, RNAi-mediated silencing of irc2/bf inhibited phagocytosis of B. afzelii and C. albicans but not the other bacteria. Tissue expression profiles, specific responses to microbial challenges, and patterns of phagocytic phenotypes upon RNAi silencing observed for IrC2/Bf match well with the previously reported characteristics of I. ricinus C3-related molecule 1 (IrC3-1). Therefore we presume that IrC2/Bf functions as a convertase in the same complement activation pathway protecting ticks against yeast and Borrelia infection.

Quantitative and microscopic tracking of Borrelia afzelii transmission from infected Ixodes ricinus nymphs has shown a transmission cycle different from that of Borrelia burgdorferi and Ixodes scapularis. Borrelia afzelii organisms are abundant in the guts of unfed I. ricinus nymphs, and their numbers continuously decrease during feeding., Quantitative and microscopic tracking of Borrelia afzelii transmission from infected Ixodes ricinus nymphs has shown a transmission cycle different from that of Borrelia burgdorferi and Ixodes scapularis. Borrelia afzelii organisms are abundant in the guts of unfed I. ricinus nymphs, and their numbers continuously decrease during feeding. Borrelia afzelii spirochetes are present in murine skin within 1 day of tick attachment. In contrast, spirochetes were not detectable in salivary glands at any stage of tick feeding. Further experiments demonstrated that tick saliva is not essential for B. afzelii infectivity, the most important requirement for successful host colonization being a change in expression of outer surface proteins that occurs in the tick gut during feeding. Spirochetes in vertebrate mode are then able to survive within the host even in the absence of tick saliva. Taken together, our data suggest that the tick gut is the decisive organ that determines the competence of I. ricinus to vector B. afzelii. We discuss possible transmission mechanisms of B. afzelii spirochetes that should be further tested in order to design effective preventive and therapeutic strategies against Lyme disease.

Background Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year. Results We have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species. Fil: Kosakyan, Anush. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Alama Bermejo, Gema. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; Argentina. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. State University of Oregon; Estados Unidos Fil: Bartošová-Sojková, Pavla. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Born-Torrijos, Ana. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Šíma, Radek. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Nenarokova, Anna. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. University of South Bohemia; República Checa Fil: Eszterbauer, Edit. Hungarian Academy Of Sciences; Hungría Fil: Bartholomew, Jerri. State University of Oregon; Estados Unidos Fil: Holzer, Astrid Sybylle. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa

Quantitative tracking ofBorrelia afzeliihas shown that its transmission cycle differs from the salivary route ofB. burgdorferitransmission byIxodes scapularis.Borrelia afzeliiare abundant in the guts of unfedIxodes ricinusnymphs and their numbers continuously decrease during feeding. In contrast, spirochetes are not present in the salivary glands.Borrelia afzeliitransmission starts during the early stages of feeding, spirochetes could be detected in murine skin within 1 day of tick attachment. Tick saliva is not essential forB. afzeliiinfectivity, the main requirement for successful host colonization being a change in outer surface protein expression that occurs in the tick gut during feeding. Spirochetes in vertebrate mode are able to survive within the host even if the tick is not present. On the basis of our data we propose that a direct ‘gut-to-mouth’ route of infection appears to be the main route ofB. afzeliitransmission.ImportanceLyme borreliosis is the most common vector-borne disease in the USA and Europe. The disease is caused by theBorreliaspirochetes and is transmitted throughIxodesticks. A better understanding of howBorreliaspirochetes are transmitted is crucial for development of efficient vaccines for preventing Lyme borreliosis. Here we present that the transmission of EuropeanB. afzeliispirochetes byI. ricinusticks significantly differs from the model transmission cycle described for AmericanB. burgdorferi/I. scapularis. We suggest thatB. afzeliiis not transmitted via salivary glands but most likely through the ‘midgut to mouthpart’ route. We further demonstrate that tick saliva is not important forB. afzeliitransmission and infectivity. Therefore, we support early studies by Willy Burgdorfer, who proposed thatBorreliatransmission occurs by regurgitation of infected gut contents. Our findings collectively point to theBorrelia-tick midgut interface as the correct target in our endeavours to combat Lyme borreliosis.

Background Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. Results We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. Conclusions Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species. Electronic supplementary material The online version of this article (doi: 10.1186/s13071-017-2579-5) contains supplementary material, which is available to authorized users.

Ticks are important vectors of serious human and animal disease-causing organisms, but their innate immunity can fight invading pathogens and may have the ability to reduce or block transmission to mammalian hosts. Lectins, sugar-binding proteins, can distinguish between self and non-self-oligosaccharide motifs on pathogen surfaces. Although tick hemolymph possesses strong lectin activity, and several lectins have already been isolated and characterized, little is known about the implementation of these molecules in tick immunity. Here, we have described and functionally characterized fibrinogen-related protein (FReP) lectins in Ixodes ticks. We have shown that the FReP family contains at least 27 genes (ixoderins, ixo) that could, based on phylogenetic and expression analyses, be divided into three groups with differing degrees of expansion. By using RNA interference-mediated gene silencing (RNAi) we demonstrated that IXO-A was the main lectin in tick hemolymph. Further, we found that ixoderins were important for phagocytosis of Gram-negative bacteria and yeasts by tick hemocytes and that their expression was upregulated upon injection of microbes, wounding, or after blood feeding. However, although the tick hemocytes could swiftly phagocytose Borrelia afzelii spirochetes, their transmission and burst of infection in mice was not altered. Our results demonstrate that tick ixoderins are crucial immune proteins that work as opsonins in the tick hemolymph, targeting microbes for phagocytosis or lysis.

Helminths and bacteria are major players in the mammalian gut ecosystem and each influences the host immune system and health. Declines in helminth prevalence and bacterial diversity appear to play a role in the dramatic rise of immune mediated inflammatory diseases (IMIDs) in western populations. Helminths are potent modulators of immune system and their reintroduction is a promising therapeutic avenue for IMIDs. However, the introduction of helminths represents a disturbance for the host and it is important to understand the impact of helminth reintroduction on the host, including the immune system and gut microbiome. We tested the impact of a benign tapeworm, Hymenolepis diminuta, in a rat model system. We find that H. diminuta infection results in increased interleukin 10 gene expression in the beginning of the prepatent period, consistent with induction of a type 2 immune response. We also find induction of humoral immunity during the patent period, shown here by increased IgA in feces. Further, we see an immuno-modulatory effect in the small intestine and spleen in patent period, as measured by reductions in tissue immune cells. We observed shifts in microbiota community composition during the patent period (beta-diversity) in response to H. diminuta infection. However, these compositional changes appear to be minor; they occur within families and genera common to both treatment groups. There was no change in alpha diversity. Hymenolepis diminuta is a promising model for helminth therapy because it establishes long-term, stable colonization in rats and modulates the immune system without causing bacterial dysbiosis. These results suggest that the goal of engineering a therapeutic helminth that can safely manipulate the mammalian immune system without disrupting the rest of the gut ecosystem is in reach.

The present concept of the transmission of Lyme disease from Borrelia-infected Ixodes sp. ticks to the naïve host assumes that a low number of spirochetes that manage to penetrate the midgut epithelium migrate through the hemocoel to the salivary glands and subsequently infect the host with the aid of immunomodulatory compounds present in tick saliva. Therefore, humoral and/or cellular immune reactions within the tick hemocoel may play an important role in tick competence to act as a vector for borreliosis. To test this hypothesis we have examined complement-like reactions in the hemolymph of the hard tick Ixodes ricinus against Borrelia afzelii (the most common vector and causative agent of Lyme disease in Europe). We demonstrate that I. ricinus hemolymph does not exhibit borreliacidal effects comparable to complement-mediated lysis of bovine sera. However, after injection of B. afzelii into the tick hemocoel, the spirochetes were efficiently phagocytosed by tick hemocytes and this cellular defense was completely eliminated by pre-injection of latex beads. As tick thioester-containing proteins (T-TEPs) are components of the tick complement system, we performed RNAi-mediated silencing of all nine genes encoding individual T-TEPs followed by in vitro phagocytosis assays. Silencing of two molecules related to the C3 complement component (IrC3-2 and IrC3-3) significantly suppressed phagocytosis of B. afzelii, while knockdown of IrTep (insect type TEP) led to its stimulation. However, RNAi-mediated silencing of T-TEPs or elimination of phagocytosis by injection of latex beads in B. afzelii-infected I. ricinus nymphs had no obvious impact on the transmission of spirochetes to naïve mice, as determined by B. afzelii infection of murine tissues following tick infestation. This result supports the concept that Borrelia spirochetes are capable of avoiding complement-related reactions within the hemocoel of ticks competent to transmit Lyme disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ceratomyxa puntazzi affects the sharpsnout seabream, Diplodus puntazzo , a recently explored aquaculture species in the Mediterranean. Little is known about the transmission and seasonality of marine myxozoans, although this knowledge is of considerable importance for the design of management strategies for aquaculture. In the present study on C. puntazzi we investigated the potential pathways of transmission as well as the parasite abundance in fish and its density in environmental water samples, throughout a full year. We performed monthly sentinel fish exposures in a C. puntazzi enzootic environment and quantified waterborne stages in seawater. Two novel C. puntazzi -specific PCR and quantitative PCR assays were developed to determine infection levels in fish and water samples. Ceratomyxa puntazzi presents marked seasonal changes in parasite density, with a double-peaked prevalence of infection in sentinel fish in spring and late summer/autumn, at 16–24 °C, and a covert infection during the winter months. Invasive blood stages were detected all year round by PCR. The combination of sentinel fish exposure with the quantification of waterborne stages allowed us to attribute this pattern in C. puntazzi density to higher numbers of actinospores in the water, while myxospores are predominant in summer and winter. We demonstrated that temperature increase triggered actinospore production in the invertebrate host in a benthic habitat and we suggest that the life cycle dynamics of the invertebrate host explain the double-peaked infection prevalence in fish. Experimental transmission of different C. puntazzi developmental stages in seawater or by oral and intracoelomic injection was unsuccessful which indicates fish-to-fish transmission is unlikely to occur in aquaculture systems. This is the first model studying seasonality and infection dynamics of a marine myxozoan.

A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group.Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory.Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods.The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Haem and iron homeostasis in most eukaryotic cells is based on a balanced flux between haem biosynthesis and haem oxygenase-mediated degradation. Unlike most eukaryotes, ticks possess an incomplete haem biosynthetic pathway and, together with other (non-haematophagous) mites, lack a gene encoding haem oxygenase. We demonstrated, by membrane feeding, that ticks do not acquire bioavailable iron from haemoglobin-derived haem. However, ticks require dietary haemoglobin as an exogenous source of haem since, feeding with haemoglobin-depleted serum led to aborted embryogenesis. Supplementation of serum with haemoglobin fully restored egg fertility. Surprisingly, haemoglobin could be completely substituted by serum proteins for the provision of amino-acids in vitellogenesis. Acquired haem is distributed by haemolymph carrier protein(s) and sequestered by vitellins in the developing oocytes. This work extends, substantially, current knowledge of haem auxotrophy in ticks and underscores the importance of haem and iron metabolism as rational targets for anti-tick interventions. DOI: http://dx.doi.org/10.7554/eLife.12318.001, eLife digest Ticks are small blood-feeding parasites that transmit a range of diseases through their bites, including Lyme disease and encephalitis in humans. Like other blood-feeders, ticks acquire essential nutrients from their host in order to develop and reproduce. Iron and haem (the iron-containing part of haemoglobin) are essential for the metabolism of every breathing animal on Earth. Most organisms obtain iron by degrading haem and, reciprocally, most of the iron in cells is used to make haem. However, an initial search of existing genome databases revealed that ticks lack the genes required to make the proteins that make and degrade haem. Perner et al. wanted to find out if ticks can steal haem from the host and use it for their own development. To achieve this, Perner et al. exploited a method of tick membrane feeding that simulates natural feeding on a host by using a silicone imitation of a skin and cow smell extracts (“l´odeur de vache”). Ticks were fed either a haemoglobin-rich (whole blood) or a haemoglobin-poor (serum) diet. This experiment revealed that ticks can develop normally without haemoglobin, but female ticks fed a haemoglobin-poor diet lay sterile eggs out of which no offspring can hatch. Further investigation showed that haemoglobin is vitally important as a source of haem but not as a source of the amino acids needed to produce the vitellin proteins that nourish embryos. As ticks are not armed with the ability to degrade haem, they do not acquire iron from the host haem but rather from a serum transferrin, a major iron transporter protein found in mammalian blood. Further experiments revealed that ticks have evolved proteins that can transport and store haem and so make the obtained haem available across the whole tick body. Overall, Perner et al.’s findings suggest that targeting the mechanisms by which ticks metabolise haem and iron could lead to the design of new “anti-tick” strategies. DOI: http://dx.doi.org/10.7554/eLife.12318.002

A cohort of a heretofore not described rare subtype of renal oncocytoma, small cell oncocytoma with pseudorosettes is presented. Patients were 6 women and 4 men with ages ranging from 51 to 76 years. The tumors displayed areas composed of small cells ("oncoblasts") featuring scant cytoplasm and small, round monomorphic nuclei. The small cell areas constituted 15% to 60% of the total tumor volume (mean, 28.5%; median, 22.5%). No necrosis or mitotic activity was discerned. All tumors also contained areas composed of characteristic oncocytes comprising 40% to 85% of the total tumor volume. In all cases, a varying number of pseudorosettes were identified. The pseudorosettes were composed of small globules of (periodic acid-Schiff-positive) hyaline basal membrane-like material surrounded by small "oncoblastic" cells. The immunohistochemical profile was variable, including at least focal positivity for AE1-3 (10/10), cytokeratin 7 (7/10), epithelial membrane antigen (10/10), c-kit (6/10), antimitochondrial antigen (MIA;10/10), PAX-2 (9/10), AMACR (racemase;6/10), CD10 (5/10), parvalbumin (8/10), vimentin (6/10), claudin 7 (10/10), and claudin 8 (3/10). No immunoreactivity for carbonic anhydrase 9, HMB-45, S-100A1, and TFE3 was documented. We found no differences in the immunophenotype in the small cell oncocytes/oncoblasts that formed pseudorosettes and those that did not. However, there were differences in the immunohistochemical profile of classic oncocytes and small cell oncocytes/oncoblasts. Using array comparative genomic hybridization, no chromosomal changes were identified in any of the cases examined (n = 3). No numerical changes of chromosomes 7 and 17 were revealed on fluorescence in situ hybridization analysis (n = 3). In conclusion, we herein present the first study on small cell renal oncocytomas with formation of pseudorosettes. This is a rare subtype of oncocytoma, which may, especially on a core biopsy, present differential diagnostic difficulties. The immunohistochemical profile of these tumors is variable and differs in significant respects from that of conventional renal oncocytoma. Awareness of this entity and its immunohistochemical variability should help in distinguishing this rare tumor from malignant tumors with similar (small cell) histomorphologic features. All tumors behaved in a benign fashion during follow-up (mean, 3.1 years; median, 1 year).

We report a case of an extra nuchal-type fibroma in a 51-year-old male suspected to have attenuated familial adenomatous polyposis (Gardner's syndrome), who presented with a longstanding buttock mass excised due to enlargement and pain. Histopathologically, lobules of haphazard, hypocellular, hyalinized collagen bundles replaced the dermis and subcutis and entrapped nerve bundles, mimicking Morton neuroma. Ramifying nerve twigs found around larger nerve fascicles showed the co-existence of traumatic neuroma. Elastic tissue stain revealed elastosis characterized by large, arborizing fibers lying between and within the hyalinized collagen bundles. Modified Masson's trichrome stain showed light blue staining of collagen bundles producing the hyalinized nodules with irregular, light red staining of collagen bundles at their periphery and within tumor collagen. Compression and/or degeneration of collagen and secondary elastosis with later entrapment by tumor collagen could explain this microscopic phenotype. By immunohistochemistry, tumor spindle cells expressed nuclear β-catenin and cyclin D1, mostly within regions of fibrosis implicating activation of the adenomatous polyposis coli (APC)-Wnt pathway. Genetic analysis showed a missense mutation in APC gene (c.7504G>A, p.G2502S in exon 15) and a functional homozygous polymorphism in the MUTYH gene (c.36+325G>C, (IVS1+5G/C)). Nuchal-type fibroma has been associated with Gardner's syndrome and trauma. In this patient, genetic predisposition coupled with repetitive, localized trauma and collagen degeneration may have provided the stimulus for the development of extra nuchal-type fibroma.

Recently, renal angiomyoadenomatous tumor (RAT) has been identified. However, there are no descriptions about clear cell renal cell carcinoma (RCC) with focal RAT-like features. A 33-year-old Japanese man was found to have a tumor in the left kidney. Macroscopically, the tumor extended into the perinephric fat tissue, and the cut surface showed the yellowish color. The histologic examination of the tumor consisted of 2 components of clear cell RCC and RAT-like area. The RAT-like area showed the admixture of epithelial cells with basophilic or clear cytoplasm and stromal component containing leiomyomatous stroma, fine capillary network, and pericytic network. Immunohistochemically, epithelial neoplastic cells in RAT-like area were diffusely positive for CD10 and RCC Ma. G-band karyotype showed the structural abnormality of chromosome 3 and both components of clear cell RCC and RAT-like area revealed the identical VHL gene mutation. Finally, pathologists should pay attention to the presence of clear cell RCC focally resembling RAT.

Few genetic studies of renal carcinoid tumor have been conducted thus far. We performed immunohistochemical and genetic examinations on four renal carcinoid tumors. Histologically, the tumors consisted of neoplastic cells with round to oval nuclei. Various growth patterns such as tightly packed cords and trabeculae, ribbon-like, trabecular, sheet-like or solid growth were observed. Nuclear chromatin showed a coarse and granular pattern. Immunohistochemically, tumors were positive for chromogranin A and synaptophysin. In the fluorescence in situ hybridization study, three of four tumors revealed monosomy of chromosome 3 (D3Z1), but one tumor showed monosomy of chromosome 13 (D13S319/13q34). Using PCR amplification and fragment analysis of three microsatellite markers (D3S1300, D3S666 and D3S1768) of chromosome arm 3p, one tumor showed loss of heterozygosity at D3S1300 and D3S1768, one tumor was not informative and the analysis of two tumors failed due to low DNA quality. In three cases, the VHL gene status was tested. Two tumors showed wild-type, but the analysis of one tumor failed to provide adequate results. In conclusion, we suggest that the abnormality of chromosome 3 is involved in the pathogenesis of renal carcinoid tumor.

We present a series of a distinct tumorous entity named renal angiomyoadenomatous tumor (RAT). Five cases were retrieved from the consultation files of the authors. Histologic and immunohistochemical features were evaluated. Sequencing analysis of coding region of the VHL gene was carried out in all cases. The tumors were composed of admixture of an epithelial clear cell component and prominent leiomyomatous stroma. Epithelial cells formed adenomatous tubular formations endowed with blister-like apical snouts. All tubular/glandular structures were lined by a fine capillary network. The epithelial component was positive for epithelial membrane antigen, CK7, CK20, AE1-AE3, CAM5.2, and vimentin in all cases. In all analyzed samples, no mutation of the VHL gene was found. RAT is a distinct morphologic entity, being different morphologically, immunohistochemically, and genetically from all renal tumors including conventional clear cell carcinoma and mixed epithelial and stromal tumor of kidney.

Endolymphatic sac tumor (Heffner tumor) (ELST) is a very rare nonmetastasizing, locally aggressive low-grade adenocarcinoma of endolymphatic sac origin, which is linked to von-Hippel-Lindau disease (VHLD). VHLD is an autosomal dominant disorder characterized by an inherited genetic abnormality of the VHL gene located on the short arm of chromosome 3 (3p26-p25). VHL gene mutations have been shown both in ELSTs associated with VHLD and in sporadic cases. Because of the rarity of ELST, only a small number of cases have been subjected to molecular genetic analysis. We have encountered two patients with ELST, one of whom presented with a medical and family history of VHLD. The second was a sporadic case, the patient having no symptoms of VHLD. The tissues obtained from Heffner tumor and cerebellar hemangioblastoma from the patient with inherited VHLD possess a point mutation in exon 1 of VHL gene. This mutation is a C to T exchange at position 194, resulting in amino acid exchange S65L. No mutation was found in any of the three exons analyzed and in the exon-intron junctions of the VHL gene in the sporadic case.

Although prominent vascular proliferation is a known feature of various neuroendocrine tumors, it has not been systematically studied in Merkel cell carcinoma (MCC) of the skin. The purpose of this study was to fully characterize the light microscopic, immunohistochemical, and ultrastructural features of vascular changes associated with MCC and to determine their frequency and differential diagnostic implications. Additionally, the presence of human herpesvirus 8 DNA in the lesional tissue was investigated. Of 92 studied cases of MCC, 18 cases (20%) were found to exhibit foci of prominent vascular changes which were classified into the following 6 patterns: pericyte hyperplasia, pyogenic granuloma-like, hemangioendothelioma-like, epithelioid hemangioma-like, peliosis-like, and follicular dendritic cell tumor-like pattern. In addition, Azzopardi phenomenon was observed. These changes occurred singly or in combination. Human herpesvirus 8 DNA was identified by polymerase chain reaction in none of the 18 cases. It is concluded that prominent vascular proliferations may be seen in 20% of MCC, and thereby, MCC resembles neuroendocrine tumors in other organs. When unduly prominent and encountered in a limited biopsy specimen, vascular alterations may represent a potential diagnostic pitfall, but, on the other hand, they themselves may serve as a clue to the correct diagnosis. Human herpesvirus 8 does not play a role in angiogenesis in MCC.

We attempted to investigate the clinicopathological correlation of renal oncocytoma (RO) with renal vein extension. We identified seven ROs with extension into the branches of renal vein. The age of seven patients ranged from 61 to 82 years. Five cases were identified incidentally; two patients had gross hematuria. After surgery, all patients were alive and free of tumors with follow-up of 1 to 5 years (mean = 3.6). Oncocytomas measured from 2.2 to 7.5 cm. Renal vein extension was grossly suspected in five of seven cases and histologically confirmed in all seven cases. Tumor cells were positive for cytokeratins, mitochondrial-antigen (MIA), epithelial membrane antigen (EMA), and parvalbumin; five of seven tumors were focally positive for CD117. Ultrastructurally, the cytoplasm was packed by mitochondria. Molecular genetic analysis did not detect abnormal numbers of chromosomes 1, 2, 6, 7, 10, 17, and XY by fluorescence in situ hybridization, loss of heterozygosity on 3p and mutation of von Hippel-Lindau gene in all cases. Array comparative genomic hybridization analysis of two cases did not show any major genetic changes. Our conclusions are as follows: (1) renal oncocytomas may have intravascular extension to the branches of the renal vein; (2) renal oncocytomas with intravascular extension to the branches of the renal vein have the same morphological, immunohistochemical, and cytogenetic findings as have their counterparts without evidence of intravascular invasion; (3) the absence of metastases suggests an overall benign behavior of this tumor, but this has to be substantiated by further studies with a long-term follow-up; and (4) in a renal tumor with granular cytoplasm showing renal vein extension, it is necessary to carefully exclude renal cell carcinomas such as chromophobe RCC, oncocytic variant of papillary RCC, and granular variant of clear cell RCC.

The authors describe 2 tumors that, to the best of their knowledge, are hitherto undescribed. The predominant cell type was small round to fusiform dark blue cells. The dark blue cells formed distinct epithelial cords with gland-like formations with mucicarmine-positive mucus. Another distinctive component of the tumors was a mesenchymal one. The mesenchymal areas appeared benign and could be likened to a fibroma having a densely collagenous stroma, or they had spindle cells set in the myxoid background, rendering a myxoma-like appearance. Another distinctive feature was ganglion cell differentiation. Mitotic figures, including atypical forms, were found only in the small cell component. All cells were immunohistochemically negative for actin, calponin, desmin, HMB45, neurofilament protein, CD99/MIC2, Melan A, tyrosinase, serotonin, CD56, Melan A, GFAP, and S-100 protein. Cytokeratin, synaptophysin, FLI1 protein, and chromogranin antibodies reacted only in the primitive small round cells, while all the other components were cytokeratin negative. Fluorescence in situ hybridization showed that the tumors are without the EWSR1 gene translocation and gain 12p. Ultrastructurally, the cells were endowed with well-formed intercellular desmosomes membrane-bound secretory in the cytoplasm. Granules were found in the cytoplasm. We suggest the name “primitive small cell tumor with epithelial, gangliocytic, neuroendocrine, and mesenchymal differentiation” for this neoplasm.

The tick species Ixodes uriae , parasitizing seabirds in the Arctic, may transmit many pathogens including various arboviruses, Borrelia spirochetes and Babesia apicomplexans. These pathogens may pose an important additional stress to seabirds, which are already stressed by environmental changes such as pollutants and decreased food availability. Here, we present the results of the first screening for arboviruses of the genera Flavivirus, Alphavirus, Orthobunyavirus, Phlebovirus and Orbivirus, as well as Borrelia spirochetes and Babesia apicomplexans from Svalbard and Jan Mayen. Using polymerase chain reaction technology with genus-specific primers, we tested 89 ticks collected on Jan Mayen, Bjornoya and Spitsbergen between 2008 and 2012. We did not detect any of the screened tick-borne pathogens. Nevertheless, these pathogens may be introduced to Svalbard and Jan Mayen by migratory birds in the near future. The increasing numbers of ticks appearing in the studied areas make this introduction even more likely. Such an introduction would have serious impact on seabird ecology as well as on human public health. Therefore, continuous careful surveillance and monitoring of possible tick-borne pathogen introductions is important. Keywords: Tick; Ixodes uriae ; tick-borne pathogens; arboviruses; Borrelia spirochetes; Babesia apicomplexans. (Published: 20 October 2015) To access the supplementary material for this article, please see supplementary files in the column to the right (under Article Tools). Citation: Polar Research 2015, 34 , 27466, http://dx.doi.org/10.3402/polar.v34.27466

Ticks are blood-feeding parasites and vectors of serious human and animal diseases. Ixodes ricinus is a common tick in Europe, transmitting tick-borne encephalitis, Lyme borreliosis, anaplasmosis, or babesiosis. Immunization of hosts with recombinant tick proteins has, in theory, the potential to interfere with tick feeding and block transmission of pathogens from the tick to the host. However, the efficacy of tick antigens has, to date, not been fully sufficient to achieve this. We have focused on 11 in silico identified genes encoding proteins potentially involved in tick iron and heme metabolism. Quantitative real-time PCR (qRT-PCR) expression profiling was carried out to preferentially target proteins that are up-regulated during the blood meal. RNA interference (RNAi) was then used to score the relative importance of these genes in tick physiology. Finally, we performed vaccination screens to test the suitability of these proteins as vaccine candidates. These newly identified tick antigens have the potential to improve the available anti-tick vaccines.

We present a case of an unusual tumor that occurred in the perianal area of a 64-year-old woman. Clinical investigation revealed no tumor elsewhere. The lesion was removed and the patient is alive without signs of metastasis or recurrence 5.5 years after surgery. Histopathologically, the neoplasm was composed of single-cell cords of uniform round to ovoid cells intermixed with round to elongated tubules showing decapitation secretion at the luminal border. The tubules were mainly composed of a single cell layer, but focally multilayered epithelium (without evidence of myoepithelial cell differentiation) was seen as well as discrete cribriform structures and intraluminal bridges. Overall, the cell cord component slightly dominated over the tubular component, and the two were intermixed. A vague targetoid arrangement of the cell cords was seen focally. Immunohistochemically, the tumor cells in both components reacted positively for E-cadherin, 34betaE12, estrogen receptors and progesterone receptor and were negative for HER2/neu (c-erbB-2). There was no evidence of myoepithelial cell differentiation with calponin. We believe that the present case is best classified as mammary type tubulolobular carcinoma and, given the location, the origin in anogenital mammary-like glands most likely.

Sclerosing polycystic adenosis (SPA) is a recently described, rare lesion of the salivary glands that bears a resemblance to epithelial proliferative lesions of the breast. The true nature of the lesion is unknown, but up to now it has been generally believed to represent a pseudoneoplastic sclerosing and inflammatory process. However, local recurrence developed in about one-third of the cases. Superimposed dysplastic changes ranging from low-grade dysplasia to carcinoma in situ were described in SPA. Although no metastases-related and/or disease-related patient deaths were documented, these clinical and histopathologic features raise the possibility that SPA might represent a neoplastic lesion. Polymorphism of the human androgen receptor locus is most frequently used to assess whether the pattern of X-chromosome inactivation is random or nonrandom, the latter strongly indicating clonality. In this study, the assay was applied to tissue from 12 examples of SPA. Three cases (males) were noninformative and 3 cases (females) could not be analyzed owing to poor quality of DNA, but all the remaining 6 lesions satisfied the criteria for monoclonality. We therefore conclude that the findings in the present study are further supporting evidence that SPA is a neoplasm, and not just a reactive process.

In mammary pathology, adenosis tumor is defined as a clinically recognizable lesion that histologically primarily consists of adenosis, but also exhibits various combinations of diverse epithelial changes seen in other benign breast diseases. A lesion that occurred in the anogenital area of a 46-year-old woman and apparently arose in anogenital mammary-like glands is described and which, in our opinion, is best classified as adenosis tumor. A biopsy revealed a well-demarcated, unencapsulated lesion surrounded by compressed fibrous tissue forming a pseudocapsule. Several histological patterns within the same tumor mass were recognizable: sclerosing adenosis-like changes, variably sized microcysts and cysts, some with rare short papillary projections having hyalinized cores, rare tubular structures exhibiting epithelial features reminiscent of simple ductal hyperplasia, areas with oxyphilic (apocrine) metaplasia, and clear cell epithelial changes resembling mucinous metaplasia. Decapitation secretion was notable in many lumens. Rare lumens were filled with foamy macrophages. There were also focal clear cell changes of myoepithelial cells. The stroma was paucicellular and sclerotic in some foci and composed of myofibroblasts and myxoid in others. Calponin, actins, and p63 stained myoepithelial cells. The cells in the oxyphilic (apocrine) metaplasia areas stained for mitochondrial antigen and Bcl-2. Antibodies to progesterone and estrogen receptor stained approximately 50 and 20% of the epithelial cell population, respectively. Human androgen receptor gene analysis yielded a monoclonal pattern. As our case exhibited a number of patterns identical to those seen in diverse benign breast diseases, its classification as adenosis tumor seems justifiable. This cutaneous perianal lesion is indistinguishable microscopically from its mammary analogue and was clinically detectable.

A hitherto unrecognized variant of solid-pseudopapillary neoplasm of the pancreas is reported. The tumor presented in the pancreatic head of a 57-year-old female patient. It was a well-circumscribed, encapsulated nodule measuring 27 mm in diameter, with variegated yellow to brown and gray cut surface. Histologically, the neoplasm was composed of uniform polyhedral cells arranged around delicate fibrovascular cores retaining their solid pattern in the periphery, whereas central parts of the tumor were characterized by the formation of papillae and smaller pseudocysts. Neither mitotic activity nor invasive growth were found. Immunohistochemically, tumor cells were positive for vimentin, neuron-specific enolase, and CD56, whereas they were negative in reactions with antibodies directed against other neuroendocrine markers, cytokeratins, melanocytic markers, and pancreatic amylase. In addition to these typical findings, intracellular pigmented granules were found in the darker brown zones of the tumor. They were positively stained in periodic acid-Schiff reaction after diastase digestion, sudan black B, and in Schmorl stain. In contrast, they were not stained with Fontana-Masson, Ziehl-Neelsen, and Perls stains. Ultrastructurally, the pigment consisted of dense granules with lipid droplets resembling modified lysosomes. These results exclude the possibility of a melanogenic nature of the pigment and instead determine it as lipofuscin.

A unique and never-before-published example of clear cell renal cell carcinoma (CRCC) found among 9000 primary renal cell tumors is presented. The tumor contained such a dense concentration of glassy hyaline globules (GHG) in the cytoplasm of the neoplastic cells that it overshadowed the morphology of the neoplasm. The resulting appearance of the tumor was quite misleading and different from the conventional CRCC. The GHG were 7-30 microm in diameter. They were glassy and pale to slightly eosinophilic in color in hematoxylin-eosin. The GHG stained positively with periodic acid-Schiff and negatively with silver and Hale's colloidal iron. Immunohistochemically, they were also negative for anti Mycobacterium bovis antigen. At the ultrastructural level, the GHG were formed by an amorphous mass of stellate shape. The GHG were localized within rough endoplasmic reticulum. Molecular genetically, mutation of the distal part of exon 3 of the VHL gene causing elongation of the VHL protein was found in the tumor, thus confirming the diagnosis of CRCC.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor- SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found., This research was supported by grants BFU2011-23896 and the European Union FP7 ANTIGONE project number 278976 (JdlF). NA was funded by Ministerio de Educacion y Ciencia, Spain. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007–2013), Spain. RS was supported by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and by the Grant 13-12816P (GA CR). OH was supported by the Grant Agency of the Czech Republic grant no. 13-27630P and European Union FP7 MODBIOLIN project number 316304. CR and LBS were supported by the United Kingdom Biotechnology and Biological Sciences Research Council's National Capability Grant to the Pirbright Institute.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi. [Methods]: Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted. [Results]: Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC value: ∼1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18. [Conclusion]: Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis., Miray Tonk is a Marie Curie Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). This project was partially supported by the Grant Agency of the Czech Republic (GACR P302/11/1901) and with institutional support RVO: 60077344 from Biology Centre, Institute of Parasitology as well as grant ANTIGONE (EU-7FP; 278976). James J. Valdés was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic. Radek Šíma was supported by the Grant 13-12816P (GA CR). Mohammad Rahnamaeian and Andreas Vilcinskas acknowledge the Ministry for Science and Art of the State of Hesse (Germany) for funding the LOEWE Center of Insect Biotechnology and Bioresources. Zdeněk Franta was supported by Alexander von Humboldt Research Fellowship for Postdoctoral Researchers.

Background: Analysis of Borrelia burgdorferi ospC types from the southeastern U.S.A. supported the common belief that various ospC types are geographically restricted and host specific. Being widely distributed in the region, the southeastern population of B. burgdorferi is represented by a surprisingly small number of ospC types. Types B, G and H are dominant or common and are invasive, while scarce type L, restricted mostly to the southeastern U.S.A., is believed to rarely if ever cause human Lyme disease. OspC type B and L strains are represented in the region at the same rate, however their distribution among tick vectors and vertebrate hosts is unequal. Findings: Direct diagnostics was used to analyze the ability of B. burgdorferi ospC type L strains to disseminate into host tissues. Mice were infected by subcutaneous injections of B. burgdorferi strains of various ospC types with different invasive capability. Spirochete levels were examined in ear, heart, bladder and joint tissues. Noninfected I. ricinus larvae were fed on infected mice until repletion. Infection rates were determined in molted nymphs. Infected nymphs were then fed on naive mice, and spirochete transmission from infected nymphs to mice was confirmed. Conclusions: B. burgdorferi ospC type L strains from the southeastern U.S.A. have comparable potential to disseminate into host tissues as ospC types strains commonly associated with human Lyme disease in endemic European and North American regions. We found no difference in the invasive ability of ospC type B and L strains originated either from tick vectors or vertebrate hosts.

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens.

Limulus Clotting Factor C is a multi-domain serine protease that triggers horseshoe crab hemolymph clotting in the presence of trace amounts of bacterial lipopolysaccharides. Here we describe and functionally characterize an homologous molecule, designated as IrFC, from the hard tick Ixodes ricinus. Tick Factor C consists of an N-terminal cysteine-rich domain, four complement control protein (sushi) modules, an LCCL domain, a truncated C-lectin domain and a C-terminal trypsin-type domain. Developmental expression profiling by quantitative real-time PCR revealed that the irfc mRNA is expressed in all stages including eggs. In tissues dissected from adult I. ricinus females, the irfc mRNA is present mainly in tick hemocytes and accordingly, indirect immunofluorescence microscopy localized IrFC intracellularly, in tick hemocytes. Irfc mRNA levels were markedly increased upon injection of sterile saline, or different microbes, demonstrating that the irfc gene transcription occurs in response to injury. This indicates a possible role of IrFC in hemolymph clotting and/or wound healing, although these defense mechanisms have not been yet definitely demonstrated in ticks. RNAi silencing of irfc expression resulted in a significant reduction in phagocytic activity of tick hemocytes against the Gram-negative bacteria Chryseobacterium indologenes and Escherichia coli, but not against the yeast, Candida albicans. This result suggests that IrFC plays a role in the tick primordial complement system and as such possibly mediates transmission of tick-borne pathogens.

Ixodes ricinus transmits bacterial, protozoal and viral pathogens, causing disease and forming an increasing health concern in Europe. ANTIDotE is an European Commission funded consortium of seven institutes, which aims to identify and characterize tick proteins involved in feeding and pathogen transmission. The knowledge gained will be used to develop and evaluate anti- tick vaccines that may prevent multiple human tick-borne diseases. Strategies encompassing anti-tick vaccines to prevent transmission of pathogens to humans, animals or wildlife will be developed with relevant stakeholders with the ultimate aim of reducing the incidence of tick-borne diseases in humans.

Sir, mesenchymal tumors of the pancreas are exceedingly rare, accounting for less than 1% of all pancreatic tumors, and most of them have been reported in small series or as single case reports. This group of pancreatic tumors includes leiomyosarcoma, malignant peripheral nerve sheath tumor, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma,hemangiopericytoma,schwannomaandsolitary fibrous tumor. In the gut, mesenchymal tumors are more common, and the majority of these are formed by gastrointestinal stromal tumors (GISTs). GISTs are defined as KIT-positive mesenchymal spindle cell or epithelioid neoplasms showing differentiation toward the interstitial cell of Cajal. GISTs occur most commonly in the stomach (60–70%), small intestine (20–25%), colorectum (5%) and esophagus (

Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes is likely mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia), rickettsiae (Anaplasma), and protozoans (Babesia). Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens., This work was supported by the Grant Agency of the Czech Republic (grant Nos.: 506/10/2136, 13-11043S; postdoctoral grants No. 13-27630P and No. 13-12816P). Ondrej Hajdušek is further supported by the EU FP7 project MODBIOLIN No. 316304 and Radek Šíma by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) co-financed by the European Social Fund and the state budget of the Czech Republic. The research at the Institute of Parasitology, BC ASCR is covered by RVO 60077344. This work was also supported by grants BFU2011-23896 and EU FP7 ANTIGONE project number 278976. Nieves Ayllón was funded by MEC, Spain.

Lynch syndrome (LS) is characterized by familial clustering of early cancer development in various organs. One of the histologic characteristics of carcinomas associated with LS is prominent intratumoral and peritumoral lymphocytes. The relationship between LS and the rare lymphocyte-rich clear cell renal cell carcinoma (CRCC) has been not been studied. We compared lymphocyte-rich CRCC (N=15) with CRCC with no or minimal lymphoid infiltration, occurring in young (40 y of age) patients (N=15) and in a control group of older (62 y of age) patients (N=5) with CRCC having conventional histologic features. All cases were analyzed histologically and immunohistochemically using antibodies against the mismatch repair (MMR) proteins MLH1, MSH2, MSH6, and PMS2. The mutational status of the Von Hippel-Lindau (VHL) gene was analyzed successfully in 25 cases. We found no case with complete absence of immunoreactivity for the MMR proteins/ enzymes studied in any of the groups. Hence, none of the RCCs with heavy lymphocytic infiltration displayed complete absence of protein expression of any of the MMR enzymes. We found mutation of the VHL gene in 2 of 8 tumors from group A, in 4 of 12 tumors from group B, and no mutation in the control group C. We conclude that this rare subset of renal neoplasms is not part of the spectrum of tumors seen in LS. The results of the VHL gene analysis are in concordance with published data on sporadic CRCCs.

Brooke-Spiegler syndrome (BSS) is an inherited autosomal dominant disease characterized by the development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma (cribriform trichoblastoma). BSS patients have various mutations in the CYLD gene, a tumor suppressor gene located on chromosome 16q. Our search of the literature revealed 51 germline CYLD mutations reported to date. Somatic CYLD mutations have rarely been investigated. We studied 10 patients from 8 families with BSS. Analysis of germline mutations of the CYLD gene was performed using either peripheral blood or nontumorous tissue. In addition, 19 formalin-fixed paraffin-embedded tumor samples were analyzed for somatic mutations, including loss of heterozygosity studies. A total of 38 tumors were available for histopathologic review. We have identified 8 novel germline mutations, all of which consisted of substitutions, deletions, and insertions/duplications and all except one led to premature stop codons. The substitution mutation in a single case was also predicted to disrupt protein function and seems causally implicated in tumor formation. We demonstrate for the first time that somatic events, loss of heterozygosity, or sequence mutations may differ among multiple neoplasms even of the same histologic type, occurring in the same patient.

We present a series of 16 salivary gland tumors with histomorphologic and immunohistochemical features reminiscent of secretory carcinoma of the breast. This is a hitherto undescribed and distinctive salivary gland neoplasm, with features resembling both salivary acinic cell carcinoma (AciCC) and low-grade cystadenocarcinoma, and displaying strong similarities to breast secretory carcinoma. Microscopically, the tumors have a lobulated growth pattern and are composed of microcystic and glandular spaces with abundant eosinophilic homogenous or bubbly secretory material positive for periodic acid-Schiff, mucicarmine, MUC1, MUC4, and mammaglobin. The neoplasms also show strong vimentin, S-100 protein, and STAT5a positivity. For this tumor, we propose a designation mammary analogue secretory carcinoma of salivary glands (MASC). The 16 patients comprised 9 men and 7 women, with a mean age of 46 years (range 21 to 75). Thirteen cases occurred in the parotid gland, and one each in the minor salivary glands of the buccal mucosa, upper lip, and palate. The mean size of the tumors was 2.1 cm (range 0.7 to 5.5 cm). The duration of symptoms was recorded in 11 cases and ranged from 2 months to 30 years. Clinical follow-up was available in 13 cases, and ranged from 3 months to 10 years. Four patients suffered local recurrences. Two patients died, 1 of them owing to multiple local recurrences with extension to the temporal bone, and another owing to metastatic dissemination to cervical lymph nodes, pleura, pericardium, and lungs. We have shown a t(12;15) (p13;q25) ETV6-NTRK3 translocation in all but one case of MASC suitable for analysis. One case was not analyzable and another was not available for testing. This translocation was not found in any conventional salivary AciCC (12 cases), nor in other tumor types including pleomorphic adenoma (1 case) and low-grade cribriform cystadenocarcinoma (1 case), whereas ETV6-NTRK3 gene rearrangements were proven in all 3 tested cases of mammary secretory carcinoma. Thus, our results strongly support the concept that MASC and AciCC are different entities.