Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

The Stable Matching Problem in TBEV Enzootic Circulation: How Important Is the Perfect Tick-Virus Match?

Tick-borne encephalitis virus (TBEV), like other arthropod-transmitted viruses, depends on specific vectors to complete its enzootic cycle. It has been long known that Ixodes ricinus ticks constitute the main vector for TBEV in Europe. In contrast to the wide distribution of the TBEV vector, the occurrence of TBEV transmission is focal and often restricted to a small parcel of land, whereas surrounding areas with seemingly similar habitat parameters are free of TBEV. Thus, the question arises which factors shape this focal distribution of TBEV in the natural habitat. To shed light on factors driving TBEV-focus formation, we used tick populations from two TBEV-foci in Lower Saxony and two TBEV-foci from Bavaria with their respective virus isolates as a showcase to analyze the impact of specific virus isolate-tick population relationships. Using artificial blood feeding and field-collected nymphal ticks as experimental means, our investigation showed that the probability of getting infected with the synonymous TBEV isolate as compared to the nonsynonymous TBEV isolate was elevated but significantly higher only in one of the four TBEV foci. More obviously, median viral RNA copy numbers were significantly higher in the synonymous virus-tick population pairings. These findings may present a hint for a coevolutionary adaptation of virus and tick populations.

Tick populations from endemic and non-endemic areas in Germany show differential susceptibility to TBEV.

Tick-borne encephalitis virus (TBEV) is endemic in twenty-seven European countries, transmitted via the bite of an infected tick. TBEV is the causative agent of one of the most important viral diseases of the central nervous system (CNS). In Germany, 890 human cases were registered between the years 2018-2019. The castor bean tick, Ixodes ricinus, is the TBEV vector with the highest importance in Central Europe, including Germany. Despite the nationwide distribution of this tick species, risk areas of TBEV are largely located in Southern Germany. To increase our understanding of TBEV-tick interactions, we collected ticks from different areas within Germany (Haselmühl/Bavaria, Hanover/Lower Saxony) and infected them via an in vitro feeding system. A TBEV isolate was obtained from an endemic focus in Haselmühl. In two experimental series conducted in 2018 and 2019, ticks sampled in Haselmühl (TBEV focus) showed higher artificial feeding rates, as well as higher TBEV infections rates than ticks from the non-endemic area (Hanover). Other than the tick origin, year and month of the infection experiment as well as co-infection with Borrelia spp., had a significant impact on TBEV Haselmühl infection rates. Taken together, these findings suggest that a specific adaptation of the tick populations to their respective TBEV virus isolates or vice versa, leads to higher TBEV infection rates in those ticks. Furthermore, co-infection with other tick-borne pathogens such as Borrelia spp. can lower TBEV infection rates in specific populations.

Seroprevalence and Current Infections of Canine Vector-Borne Diseases in Costa Rica.

Domestic dogs may carry several vector-borne pathogens, including zoonotic agents, especially in tropical regions like Central America. The epidemiology of these pathogens is prone to change due to urbanization, trade and travel as well as climate change, necessitating repeated monitoring. This study aims to present a comprehensive picture of canine vector-borne diseases in Costa Rica, combining data on seroprevalence with molecular species identification of the causative pathogens. In this survey, 294 dogs from all seven provinces of Costa Rica were included. After a clinical examination, diagnostic blood samples were analyzed with regard to packed cell volume (PCV) and presence of microfilaria. Serum samples were tested for antibodies against Ehrlichia spp., Anaplasma spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.) as well as antigen of Dirofilaria immitis. Seropositive and microfilaremic blood samples were analyzed by PCR to detect current infections and identify the pathogen species. Overall, 45.24% (133/294, 95% CI: 39.45-51.11%) of dogs were seropositive for at least one of the tested pathogens. Seroprevalence was highest for Ehrlichia spp. (39.46%, 116/294, 95% CI: 33.83-45.29%), followed by Babesia spp. (23.13%, 68/294, 95% CI: 18.43-28.38%), Anaplasma spp. (13.27%, 39/294, 95% CI: 9.61-17.69%), and B. burgdorferi s.l. (0.34%, 1/294, 95% CI: 0.01-1.88%). Generalized linear mixed models indicated a significant association of Ehrlichia-, Anaplasma- and Babesia-seropositivity, as well as a significant effect of age and breed on Ehrlichia-seropositivity. Furthermore, a statistically significant negative effect of Ehrlichia-, Anaplasma-, and Babesia-seropositivity on PCV was found. Regarding current infections, Ehrlichia canis DNA was detected in 51.72% (60/116, 95% CI: 42.26-61.10%) of Ehrlichia-seropositive dogs, while Ehrlichia ewingii and Ehrlichia chaffeensis were not detected. Furthermore, 10.26% (4/39, 95% CI: 2.87-24.22%) of Anaplasma-seropositive dogs were coinfected with Anaplasma phagocytophilum and Anaplasma platys, while one animal (2.56%, 95% CI: 0.65-13.48%) was infected with A. phagocytophilum only. Among Babesia-seropositive dogs, Babesia vogeli and Hepatozoon canis were detected in one animal each (1.47%, 1/68, 95% CI: 0.04-7.92%). Dirofilaria immitis antigen was detected in 4.42% (13/294, 95% CI: 2.38-7.44%) of dogs. In microfilaremic animals, D. immitis as well as Acanthocheilonema reconditum infections were identified. This survey demonstrates that canine vector-borne pathogens, including zoonotic agents like A. phagocytophilum and D. immitis, are widespread in Costa Rica. Thus, protection of dogs from disease-transmitting vectors is recommended from an animal welfare as well as public health perspective.

Draft Genome Assembly of the Poultry Red Mite, Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae, is a major worldwide concern in the egg-laying industry. Here, we report the first draft genome assembly and gene prediction of Dermanyssus gallinae, based on combined PacBio and MinION long-read de novo sequencing. The ∼959-Mb genome is predicted to encode 14,608 protein-coding genes.

Seroprevalence and current infections of canine vector-borne diseases in Nicaragua.

BACKGROUND: Vector-borne diseases constitute a major problem for veterinary and public health, especially in tropical regions like Central America. Domestic dogs may be infected with several vector-borne pathogens of zoonotic relevance, which may also severely compromise canine health. METHODS: To assess the prevalence of canine vector-borne diseases in Nicaragua, 329 dogs from seven cities, which were presented to the veterinarian for various reasons, were included in this study. Dogs were examined clinically and diagnostic blood samples were taken for analysis of packed cell volume (PCV) and presence of microfilariae as well as antigen of Dirofilaria immitis and antibodies to Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi (sensu lato) by use of a commercially available rapid ELISA. To detect current infections, specific PCRs for the detection of E. canis, A. platys and A. phagocytophilum were carried out on blood samples of the respective seropositive dogs. Microfilaremic blood samples, as well as D. immitis antigen positive samples were further subjected to PCR and subsequent sequencing for filarial species identification. RESULTS: Antibodies against Ehrlichia spp. were present in 62.9% of dogs, while Anaplasma spp. seroprevalence was 28.6%. Antibodies against species of both genera were detected in 24.9% of dogs. Borrelia burgdorferi (s.l.) antibodies were not detected. Dirofilaria immitis antigen was present in six animals (1.8%), two of which also showed D. immitis microfilariae in buffy coat. In addition to D. immitis, Acanthocheilonema reconditum was identified by PCR and sequencing in two of four additional microfilaremic blood samples, which were tested negative for D. immitis antigen. Current E. canis infections as defined by DNA detection were present in 58.5% of Ehrlichia-seropositive dogs, while 5.3% of Anaplasma-seropositive dogs were PCR-positive for A. platys, 2.2% for A. phagocytophilum and 16.0% for both Anaplasma species. Current E. canis infection had a statistically significant negative impact on PCV, whereas no relationship between infection status and clinical signs of disease could be observed. CONCLUSIONS: These results indicate that canine vector-borne diseases are widespread in Nicaragua and that dogs may constitute a reservoir for human infection with E. canis, A. phagocytophilum and D. immitis. Thus, the use of repellents or acaricides to protect dogs from vector-borne diseases is strongly recommended.

Detection of Rickettsia monacensis and Rickettsia amblyommatis in ticks collected from dogs in Costa Rica and Nicaragua.

The neotropical climate of Central America provides ideal conditions for ticks, which may transmit several human pathogens, including spotted-fever group Rickettsia. Dogs may act as sentinels or reservoirs for human tick-borne diseases due to shared tick species. Here, ticks were collected from 680 client-owned dogs in Nicaragua and Costa Rica, and a total of 316 tick pools were investigated for Rickettsia infection by quantitative real-time PCR (qPCR) targeting the gltA gene. Subsequently, up to six further genomic targets (16S rDNA, gltA, sca4, ompA, ompB and the 23S-5S intergenic spacer) were investigated for Rickettsia species determination. The predominant tick species was Rhipicephalus sanguineus sensu lato (s.l.) (19.9% of dogs infested in Costa Rica, 48.0% in Nicaragua), followed by Ixodes boliviensis (3.1% in Costa Rica / none in Nicaragua) and Amblyomma ovale (4.8% in Costa Rica, 0.9% in Nicaragua). In total, 22 of 316 tick pools containing 60 of 1023 individual ticks were Rickettsia-positive as determined by qPCR, resulting in a minimum infection rate (MIR) of 2.2%. In detail, MIR in Rh. sanguineus s.l. was 0.7% (7/281 pools), in I. boliviensis 33.3% (12/13 pools) and in A. ovale 9.7% (3/22 pools). For 11 of 12 positive I. boliviensis pools and one of six positive Rh. sanguineus s.l. pools, the species could be determined as R. monacensis. R. amblyommatis was identified in one Rh. sanguineus s.l. pool from Costa Rica and one A. ovale pool from Nicaragua. Nine of 12 R. monacensis-positive tick pools were collected in San Rafael de Heredia, Costa Rica, indicating a high local occurrence in this area. This study supports recent evidence that R. monacensis is present on the American continent. Its high local occurrence among dog-associated I. boliviensis, which may also parasitize humans, in Costa Rica gives cause for concern, as R. monacensis is also pathogenic to humans.

Genetic Characterization of Taenia saginata Cyst Isolates from Germany.

The beef tapeworm Taenia saginata, which causes taeniosis in humans and cysticercosis in cattle, is of medical and economic importance. Understanding the parasite's genetic population structure may help to analyze transmission patterns and aid in the development of control measures. As information on sequence variability is scarce for European isolates, this study aimed to elucidate the intraspecific genetic variability of T. saginata cysts from German cattle by sequence comparison of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 5 (nad5) genes, in relationship to sequences from other geographical origins. Cysts were collected from northern German, Swiss, and Belgian cattle. Moreover, proglottids from an adult T. saginata specimen from Palestine were included. Amplification and Sanger sequencing of the cox1 gene was successful for 57 samples (45 German, 9 Swiss, 2 Belgian, 1 Palestinian), whereas 32 sequences were obtained for the nad5 gene (21 German, 10 Swiss, 1 Palestinian). For German isolates, sequence comparison revealed minor genetic variability with two polymorphic sites and mutations in both genes. Three haplotypes with haplotype diversity of 0.088 for cox1 and 0.186 for nad5, as well as nucleotide diversities of 0.00028 and 0.00095, respectively, were observed. Comparison of the cox1 gene sequence of German isolates with other European, African, American, and Asian isolates obtained from National Center for Biotechnology Information (total of 71 sequences) raised 11 polymorphic sites and mutations as well as 10 haplotypes (haplotype diversity: 0.239; nucleotide diversity: 0.00097). Although nad5 sequence comparison comprised less sequences (N = 33), analyses revealed 11 polymorphic sites, 12 mutation sites, and 7 haplotypes (haplotype diversity: 0.335, nucleotide diversity: 0.00391), indicating a better resolution of genetic variability compared to cox1. Thus, nad5 may be particularly useful for in-depth studies on genetic divergence of T. saginata.

Biological function of Dictyocaulus viviparus asparaginyl peptidase legumain-1 and its suitability as a vaccine target.

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35-39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Comparison of Microbiomes between Red Poultry Mite Populations (Dermanyssus gallinae): Predominance of Bartonella-like Bacteria.

Blood feeding red poultry mites (RPM) serve as vectors of pathogenic bacteria and viruses among vertebrate hosts including wild birds, poultry hens, mammals, and humans. The microbiome of RPM has not yet been studied by high-throughput sequencing. RPM eggs, larvae, and engorged adult/nymph samples obtained in four poultry houses in Czechia were used for microbiome analyses by Illumina amplicon sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. A laboratory RPM population was used as positive control for transcriptome analysis by pyrosequencing with identification of sequences originating from bacteria. The samples of engorged adult/nymph stages had 100-fold more copies of 16S rRNA gene copies than the samples of eggs and larvae. The microbiome composition showed differences among the four poultry houses and among observed developmental stadia. In the adults' microbiome 10 OTUs comprised 90 to 99% of all sequences. Bartonella-like bacteria covered between 30 and 70% of sequences in RPM microbiome and 25% bacterial sequences in transcriptome. The phylogenetic analyses of 16S rRNA gene sequences revealed two distinct groups of Bartonella-like bacteria forming sister groups: (i) symbionts of ants; (ii) Bartonella genus. Cardinium, Wolbachia, and Rickettsiella sp. were found in the microbiomes of all tested stadia, while Spiroplasma eriocheiris and Wolbachia were identified in the laboratory RPM transcriptome. The microbiomes from eggs, larvae, and engorged adults/nymphs differed. Bartonella-like symbionts were found in all stadia and sampling sites. Bartonella-like bacteria was the most diversified group within the RPM microbiome. The presence of identified putative pathogenic bacteria is relevant with respect to human and animal health issues while the identification of symbiontic bacteria can lead to new control methods targeting them to destabilize the arthropod host.

Lyme borreliae prevalence and genospecies distribution in ticks removed from humans.

Lyme borreliosis (LB) is the most important human tick-borne disease, but Borrelia genospecies cause different clinical manifestations. Ticks of the genus Ixodes removed from humans between 2006 and 2012 were analysed for Borrelia burgdorferi sensu lato (sl) infections. The majority of ticks originated from the Greater Hanover region in Northern Germany. The engorgement status varied over the entire spectrum from unengorged (no evidence of started blood feeding) to fully engorged. In the present study, prevalence data for B. burgdorferi sl 2011 and 2012 were obtained by quantitative real-time PCR and compared to those from a former study including years 2006-2010 (Strube et al., 2011) to evaluate B. burgdorferi sl infections in ticks affecting humans over a 7-year period. In 2011, 34.2% (70/205) of adult ticks, 22.2% (94/423) of nymphs, 8.3% of larvae (1/12) as well 3 of 6 not differentiated ticks were Borrelia positive. In 2012, 31.8% (41/129) of adult ticks, 20.4% of nymphs (69/337) as well as 1 of 4 of the not differentiated ticks were determined positive. Total Borrelia infection rates decreased significantly from 23.1% in 2006 to 17.1% in 2010, followed by a significant increase to 26.0% in 2011 and 23.4% in 2012. Furthermore, B. burgdorferi sl genospecies distribution in 2006-2012 was determined in the present study by applying Reverse Line Blot technique. Borrelia genospecies differentiation was successful in 641 (67.3%) out of 953 positive tick samples. The most frequently occurring genospecies was B. afzelii (40.5% of infected ticks), followed by B. garinii/B. bavariensis (12.4%). Amongst the 641 ticks analysed for their genospecies, 74 (11.5%) carried more than one genospecies, of which 69 (10.7%) were double-infected and five (0.8%) were triple-infected. Comparison of genospecies distribution in ticks removed from humans with those from questing ticks flagged in the same geographical area revealed that ticks removed from humans were significantly more frequently infected with B. afzelii (p=0.0004), but significantly less infected with B. burgdorferi sensu stricto (p=0.0001).

Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding.

BACKGROUND: The lungworm Dictyocaulus viviparus, causing parasitic bronchitis in cattle, induces a temporary protective immunity that prevents clinical disease. A radiation-attenuated larvae based vaccine is commercially available in a few European countries, but has the disadvantages of a live vaccine. As a recombinant subunit vaccine would overcome these disadvantages, the parasite's muscle protein paramyosin (PMY) was tested as a recombinant vaccine antigen. METHODS: D. viviparus-PMY was recombinantly expressed in Escherichia coli as a glutathione-S-transferase (GST)-fused protein. Emulsified in adjuvant Saponin Quil A, the protein was given intramuscularly into calves. Two independent recombinant PMY (rPMY) vaccination trials with negative control groups (first trial: adjuvant only; second trial: non-fused GST) as well as an additional positive control group in the second trial, using the Bovilis Dictol live vaccine to verify vaccination results, were performed. To determine the vaccination success, shedding of larvae as well as worm burden and worm sizes were analyzed. Additionally, ELISA-based determination of development of immunglobulins IgM, IgA, IgE, IgG as well as the subclasses IgG1 and IgG2 was performed. To analyze PMY localization in the bovine lungworm, immunohistochemical staining of adult worms was carried out. RESULTS: Immunohistochemical staining revealed that PMY is part of the bovine lungworm's pharyngeal and body wall muscles. Vaccination with rPMY resulted in 47% [geometric mean: 67%] and 57% (geometric mean: 71%) reduction of larvae shedding in the first and second vaccination trial, respectively. Worm burden was reduced by 54% (geometric mean: 86%) and 31% (geometric mean: 68%), respectively, and worms of rPMY-vaccinated cattle were significantly shorter in both trials. Furthermore, ELISAs showed a clear antibody response towards rPMY with exception of IgE for which titers could not be detected. After challenge infection, rPMY antibodies were only exceptionally elevated among study animals indicating PMY to be a hidden antigen. CONCLUSIONS: Even though vaccination with the attenuated live vaccine was with 94% (geometric mean: 95%) reduction in larvae shedding and 93% (geometric mean: 94%) reduction in worm burden superior to rPMY vaccination, results using the latter are promising and show the potential for further development of a recombinant PMY-based vaccine against the bovine lungworm.

Whole transcriptome analysis of the poultry red mite Dermanyssus gallinae (De Geer, 1778).

SUMMARY Although the poultry red mite Dermanyssus gallinae (De Geer, 1778) is the major parasitic pest in poultry farming causing substantial economic losses every year, nucleotide data are rare in the public databases. Therefore, de novo sequencing covering the transcriptome of D. gallinae was carried out resulting in a dataset of 232 097 singletons and 42 130 contiguous sequences (contigs) which were subsequently clustered into 24 140 isogroups consisting of 35 788 isotigs. After removal of sequences possibly originating from bacteria or the chicken host, 267 464 sequences (231 657 singletons, 56 contigs and 35 751 isotigs) remained, of which 10·3% showed homology to proteins derived from other organisms. The most significant Blast top-hit species was the mite Metaseiulus occidentalis followed by the tick Ixodes scapularis. To gain functional knowledge of D. gallinae transcripts, sequences were mapped to Gene Ontology terms, Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways and parsed to InterProScan. The transcriptome dataset provides new insights in general mite genetics and lays a foundation for future studies on stage-specific transcriptomics as well as genomic, proteomic, and metabolomic explorations and might provide new perspectives to control this parasitic mite by identifying possible drug targets or vaccine candidates. It is also worth noting that in different tested species of the class Arachnida no 28S rRNA was detectable in the rRNA profile, indicating that 28S rRNA might consists of two separate, hydrogen-bonded fragments, whose (heat-induced) disruption may led to co-migration with 18S rRNA.

The predicted secretome and transmembranome of the poultry red mite Dermanyssus gallinae.

BACKGROUND: The worldwide distributed hematophagous poultry red mite Dermanyssus gallinae (De Geer, 1778) is one of the most important pests of poultry. Even though 35 acaricide compounds are available, control of D. gallinae remains difficult due to acaricide resistances as well as food safety regulations. The current study was carried out to identify putative excretory/secretory (pES) proteins of D. gallinae since these proteins play an important role in the host-parasite interaction and therefore represent potential targets for the development of novel intervention strategies. Additionally, putative transmembrane proteins (pTM) of D. gallinae were analyzed as representatives of this protein group also serve as promising targets for new control strategies. METHODS: D. gallinae pES and pTM protein prediction was based on putative protein sequences of whole transcriptome data which was parsed to different bioinformatical servers (SignalP, SecretomeP, TMHMM and TargetP). Subsequently, pES and pTM protein sequences were functionally annotated by different computational tools. RESULTS: Computational analysis of the D. gallinae proteins identified 3,091 pES (5.6%) and 7,361 pTM proteins (13.4%). A significant proportion of pES proteins are considered to be involved in blood feeding and digestion such as salivary proteins, proteases, lipases and carbohydrases. The cysteine proteases cathepsin D and L as well as legumain, enzymes that cleave hemoglobin during blood digestion of the near related ticks, represented 6 of the top-30 BLASTP matches of the poultry red mite's secretome. Identified pTM proteins may be involved in many important biological processes including cell signaling, transport of membrane-impermeable molecules and cell recognition. Ninjurin-like proteins, whose functions in mites are still unknown, represent the most frequently occurring pTM. CONCLUSION: The current study is the first providing a mite's secretome as well as transmembranome and provides valuable insights into D. gallinae pES and pTM proteins operating in different metabolic pathways. Identifying a variety of molecules putatively involved in blood feeding may significantly contribute to the development of new therapeutic targets or vaccines against this poultry pest.

Rickettsia spp. and coinfections with other pathogenic microorganisms in hard ticks from northern Germany.

Rickettsia species are the causative agent of different forms of spotted fever and thus, monitored in a number of prevalence studies. The current study examined the status of ticks from the city of Hanover, Northern Germany, regarding the presence of Rickettsia spp. and coinfections with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. In total, 1,089 questing Ixodes ricinus L. ticks were analyzed using quantitative real time polymerase chain reaction. A duplex quantitative real time polymerase chain reaction for simultaneous detection of Rickettsia spp. and Ixodes spp.-DNA as positive control for successful DNA-isolation was established. Rickettsia spp. were detected in 363 (33.3%) of the 1,089 investigated ticks. Quantification of Rickettsia showed that larvae contained up to 50,000 bacteria, nymphs up to 85 million and adults up to 200 million per tick. Species differentiation was possible in 178 out of 363 Rickettsia positive samples and resulted in a predominant occurrence of R. helvetica (98.9%, 176/178), whereas R. monacensis was rarely found (1.1%, 2/178). Besides detection of Rickettsia, positive ticks were compared with results from previous studies to examine coinfections with B. burgdorferi sl and A. phagocytophilum. The resulting coinfection rates were 9.1% (99/1,089) for B. burgdorferi sl and 2.8% (11/391) for A. phagocytophilum. Triple-infection with Rickettsia spp., B. burgdorferi sl, and A. phagocytophilum occurred in 5 (1.3%) out of 391 ticks. The current study is the first presenting quantitative data concerning the load of Ixodes ticks with Rickettsia individuals.

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany).

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany).

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.