RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection.

Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/µl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.

Epidemiological and Molecular Study on Tick-Borne Pathogens in Argun Port Area Near the Chinese-Russian Border.

Objective: We aim to investigate the species composition of ticks and the pathogen characteristics they carry in the Argun port area of the China-Russia border. Materials and Methods: Ticks were collected in surrounding grassland, mixed forest land, and other different habitats around the Argun port area at the Sino-Russian Border of Inner Mongolia in China in April 2019. The presence of 16 potential pathogens, including Yersinia Pestis, Francisella tularensis, Coxiella burnetii (Cb), Anaplasma sp. (Ap), spotted fever group rickettsiae (SFG Rk), Borrelia sp. (Bl), Leptospira, Bartonella spp., Babesia, Crimean-Congo hemorrhagic fever virus, tick-borne encephalitis virus, Bhanja virus, West Nile Virus, severe fever with thrombocytopenia syndrome bunyavirus, Hantaan virus, and bocavirus (boca) was analyzed by polymerase chain reaction. The DNA and amino acid sequences of tick-borne pathogens were compared for homology, and the phylogenetic trees were constructed by using Mega and Lasergene software. Results: A total of 210 ticks were collected and they belonged to three species: Dermacentor nuttalli, Ixodes persulcatus, and Haemaphysalis verticalis. Among them, 165 (78.57%) ticks tested positive for 5 pathogens, namely Ap, SFG Rk, Cb, Bl, and boca. Fifteen (7.14%) ticks were detected coinfection with two pathogens, and none were coinfected with three or more pathogens. Conclusion: This study shows the prevalence of at least five tick-borne pathogens in Argun, and there is a risk of coinfection by two pathogens in one tick. This study reveals the great importance of controlling tick-borne diseases in this region.

Species-Level Profiling of Ixodes pacificus Bacterial Microbiomes Reveals High Variability Across Short Spatial Scales at Different Taxonomic Resolutions.

Background and Aims: Outbreaks of severe and chronic tick-borne diseases (TBDs) are on the rise. This is through the transmission of infectious disease agents to humans during tick feeding. The transmission rate and extent of microbial exchange, however, vary based on the tick microbiome composition. While select microbes are determined to be members of the normal tick microbiome and others are clearly recognized mammalian and/or avian pathogens, the status of many other members of the tick microbiota with respect to human and alternate host pathogenesis remains unclear. Moreover, the species-level 16S microbiome of prominent TBD vectors, including Ixodes pacificus, have not been extensively studied. To elucidate the I. pacificus microbiome composition, we performed a pan-domain species-specific characterization of the bacterial microbiome on adult I. pacificus ticks collected from two regional parks within Western California. Our methods provide for characterizing nuances within cohort microbiomes and their relationships to geo-locale of origin, surrounding fauna, and prevalences of known and suspected pathogens in relation to current TBD epidemiological zones. Methods: Ninety-two adult I. pacificus bacterial microbiomes were characterized using a high-fidelity, pan-domain, species-specific, full-length 16S rRNA amplification method using circular consensus sequencing performed on the Pacific Biosciences Sequel platform. Data analyses were performed with the MCSMRT data analysis package and database. Results: The species-specific I. pacificus microbiome composition illustrates a complex assortment of microflora, including over 900 eubacterial species with high taxonomic diversity, which was revealed to vary by sex and geo-locale, though the use of full-length 16S gene sequencing. The TBD-associated pathogens, such as Borrelia burgdorferi, Anaplasma phagocytophilum, and Rickettsia monacensis, were identified along with a host of bacteria previously unassociated with ticks. Conclusion: Species-level taxonomic classification of the I. pacificus microbiome revealed that full-length bacterial 16S gene sequencing is required for the granularity to elucidate the microbial diversity within and among ticks based on geo-locale.

Genetic Analyses and Genome-Wide Association Studies on Pathogen Resistance of Bos taurus and Bos indicus Cattle Breeds in Cameroon.

Autochthonous taurine and later introduced zebu cattle from Cameroon differ considerably in their resistance to endemic pathogens with little to no reports of the underlying genetic make-up. Breed history and habitat variations are reported to contribute significantly to this diversity worldwide, presumably in Cameroon as well, where locations diverge in climate, pasture, and prevalence of infectious agents. In order to investigate the genetic background, the genotypes of 685 individuals of different Cameroonian breeds were analysed by using the BovineSNP50v3 BeadChip. The variance components including heritability were estimated and genome-wide association studies (GWAS) were performed. Phenotypes were obtained by parasitological screening and categorised in Tick-borne pathogens (TBP), gastrointestinal nematodes (GIN), and onchocercosis (ONC). Estimated heritabilities were low for GIN and TBP (0.079 (se = 0.084) and 0.109 (se = 0.103) respectively) and moderate for ONC (0.216 (se = 0.094)). Further than revealing the quantitative nature of the traits, GWAS identified putative trait-associated genomic regions on five chromosomes, including the chromosomes 11 and 18 for GIN, 20 and 24 for TBP, and 12 for ONC. The results imply that breeding for resistant animals in the cattle population from Northern Cameroon might be possible for the studied pathogens; however, further research in this field using larger datasets will be required to improve the resistance towards pathogen infections, propose candidate genes or to infer biological pathways, as well as the genetic structures of African multi-breed populations.

Epigenetic Regulation of Tick Biology and Vectorial Capacity.

Ticks exist across diverse environments and transmit numerous pathogens. Due to their long and unique life cycles, these arthropods likely evolved robust epigenetic mechanisms that provide sustainable responses and buffers against extreme environmental conditions. Herein, we highlight how the study of the epigenetic basis of tick biology and vectorial capacity will enrich our knowledge of tick-borne infections.

Phlebovirus sequences detected in ticks collected in Russia: Novel phleboviruses, distinguishing criteria and high tick specificity.

Phlebovirus is an abundant and rather heterogeneous genus within the Phenuiviridae family (order Bunyavirales). The genus Phlebovirus is divided into two antigenic complexes, which also correspond to the main vector: sandflies/mosquitoes and ticks. Previously, only sandfly/mosquito-borne phleboviruses were associated with human disease, such as Rift Valley fever virus, Toscana virus, Sicilian and Naples Sandfly fever viruses and others. Until recently, tick-borne phleboviruses were not considered as human pathogens. After the discovery of severe fever with thrombocytopenia syndrome, interest to tick-borne phleboviruses has increased dramatically. In the last decade, many novel phleboviruses have been reported in different regions. Despite this, the diversity, ecology and pathogenicity of these viruses still remain obscure. The aim of this work was to study the diversity of phleboviruses in ticks collected in several regions of Russia. We used pan-phlebovirus RT-PCR assays based on multiple degenerate primers targeting the polymerase gene fragment. Arthropod specimens were collected from 2005 to 2018. A total of 5901 Ixodidae ticks combined into 1116 pools were screened. A total of 160 specific amplicons were produced. In three cases RT-PCR assays amplified two distinct viruses from same tick pools. Direct sequencing of amplicons and subsequent phylogenetic analysis revealed twelve representatives of divergent phlebovirus groups. Based on the distribution of pairwise nucleotide sequence identity values, a cut-off (88%) was suggested to distinguish tick-borne phleboviruses. According to this provisional criterion, two viruses found here could be termed novel, while ten viruses have been described in previous studies. Detected phleboviruses demonstrated almost perfect specificity to a tick species or, at least, a genus. The same pattern was observed for tick-borne phleboviruses found in different studies around the world. Viruses that grouped together on a phylogenetic tree and differed less than this sequence identity threshold suggested above were hosted by ticks from the same genus.

The genome of Peromyscus leucopus, natural host for Lyme disease and other emerging infections.

The rodent Peromyscus leucopus is the natural reservoir of several tick-borne infections, including Lyme disease. To expand the knowledge base for this key species in life cycles of several pathogens, we assembled and scaffolded the P. leucopus genome. The resulting assembly was 2.45 Gb in total length, with 24 chromosome-length scaffolds harboring 97% of predicted genes. RNA sequencing following infection of P. leucopus with Borreliella burgdorferi, a Lyme disease agent, shows that, unlike blood, the skin is actively responding to the infection after several weeks. P. leucopus has a high level of segregating nucleotide variation, suggesting that natural resistance alleles to Crispr gene targeting constructs are likely segregating in wild populations. The reference genome will allow for experiments aimed at elucidating the mechanisms by which this widely distributed rodent serves as natural reservoir for several infectious diseases of public health importance, potentially enabling intervention strategies.

High Incidence of a Novel Rickettsia Genotype in Parasitic Haemaphysalis longicornis from China-North Korea Border.

Ticks are notorious vectors for various pathogens that cause infections in animals and humans worldwide. Rickettsia spp., a zoonotic tick-borne pathogen that could be used as a weapon agent, is widely spread in China. In the present study, ticks were collected for species identification and Rickettsia screening. PCR amplification targeting the tick 18s rRNA gene was first conducted for species validation, and then, amplification was conducted for the Rickettsia housekeeping gene for the infection rate and phylogenetic analysis. The collected ticks were identified as Haemaphysalis longicornis, 7.36% of which were Rickettsia-positive. The phylogenetic analysis showed that the Rickettsia in the parasitic ticks belonged to a novel genotype, whose closest genetic relationship was with Rickettsia heilongjiangenesis. The samples were collected in Dandong, a city on the border between China and North Korea. Considering the geographical and biological situations of the sampling sites, more extensive surveillance and risk evaluation of the tick species and tick-borne diseases are required.

Seasonal Patterns in the Prevalence and Diversity of Tick-Borne Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia spp. in an Urban Temperate Forest in South Western Slovakia.

In Europe, Ixodes ricinus is the most important vector of tick-borne zoonotic bacteria. It transmits spirochaetes from the Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum and Rickettsia spp. Although spatial differences in the prevalence of tick-borne pathogens have been intensively studied, seasonal (within-year) fluctuations in the prevalence of these pathogens within sites are often overlooked. We analyzed the occurrence and seasonal dynamics of Ixodes ricinus in an urban forest in Bratislava, Slovakia. Furthemore, we examined temporal trends in the community structure of B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in questing and bird-feeding ticks. The total prevalence for B. burgdorferi s.l. in questing I. ricinus was 6.8%, involving six genospecies with the dominance of bird-associated B. garinii and B. valaisiana.A. phagocytophilum, R. helvetica and R. monacensis occurred in 5.9%, 5.0% and 0.2% of questing ticks, respectively. In total, 12.5% and 4.4% of bird-feeding I. ricinus ticks carried B. burgdorferi s.l. and R. helvetica. The total prevalence of B. burgdorferi s.l. in our study site was two times lower than the mean prevalence for Europe. In contrast, A. phagocytophilum prevalence was significantly higher compared to those in other habitats of Slovakia. Our results imply that tick propagation and the transmission, suppression and seasonal dynamics of tick-borne pathogens at the study site were primarily shaped by abundance and temporal population fluctuations in ruminant and bird hosts.

PCR diagnosis of tick-borne pathogens in Maharashtra state, India indicates fitness cost associated with carrier infections is greater for crossbreed than native cattle breeds.

Tick-borne pathogens (TBP) are responsible for significant economic losses to cattle production, globally. This is particularly true in countries like India where TBP constrain rearing of high yielding Bos taurus, as they show susceptibility to acute tick borne disease (TBD), most notably tropical theileriosis caused by Theileria annulata. This has led to a programme of cross breeding Bos taurus (Holstein-Friesian or Jersey) with native Bos indicus (numerous) breeds to generate cattle that are more resistant to disease. However, the cost to fitness of subclinical carrier infection in crossbreeds relative to native breeds is unknown, but could represent a significant hidden economic cost. In this study, a total of 1052 bovine blood samples, together with associated data on host type, sex and body score, were collected from apparently healthy animals in four different agro-climatic zones of Maharashtra state. Samples were screened by PCR for detection of five major TBPs: T. annulata, T. orientalis, B. bigemina, B. bovis and Anaplasma spp.. The results demonstrated that single and co-infection with TBP are common, and although differences in pathogen spp. prevalence across the climatic zones were detected, simplistic regression models predicted that host type, sex and location are all likely to impact on prevalence of TBP. In order to remove issues with autocorrelation between variables, a subset of the dataset was modelled to assess any impact of TBP infection on body score of crossbreed versus native breed cattle (breed type). The model showed significant association between infection with TBP (particularly apicomplexan parasites) and poorer body condition for crossbreed animals. These findings indicate potential cost of TBP carrier infection on crossbreed productivity. Thus, there is a case for development of strategies for targeted breeding to combine productivity traits with disease resistance, or to prevent transmission of TBP in India for economic benefit.

Applying proteomics to tick vaccine development: where are we?

INTRODUCTION: Ticks are second to mosquitoes as a vector of human diseases and are the first vector of animal diseases with a great impact on livestock farming. Tick vaccines represent a sustainable and effective alternative to chemical acaricides for the control of tick infestations and transmitted pathogens. The application of proteomics to tick vaccine development is a fairly recent area, which has resulted in the characterization of some tick-host-pathogen interactions and the identification of candidate protective antigens. Areas covered: In this article, we review the application and possibilities of various proteomic approaches for the discovery of tick and pathogen derived protective antigens, and the design of effective vaccines for the control of tick infestations and pathogen infection and transmission. Expert commentary: In the near future, the application of reverse proteomics, immunoproteomics, structural proteomics, and interactomics among other proteomics approaches will likely contribute to improve vaccine design to control multiple tick species with the ultimate goal of controlling tick-borne diseases.

Genetic identification of Rickettsia sp. strain Atlantic rainforest in an endemic area of a mild spotted fever in Rio Grande do Sul state, Southern Brazil.

Rickettsia sp. strain Atlantic rainforest causes a less severe rickettsiosis, with two cases confirmed until now. The tick species Amblyomma ovale is appointed as the main vector of this bacterium. The southern region of Brazil has reported patients with spotted fever who have milder symptoms. In 2013, during an investigation of rickettsiosis cases, an A. ovale tick was found attached to a man in an area where there were two cases. The parasite was processed for molecular analysis and the rickettsial infection was confirmed based on phylogenetic analysis of genes ompA, ompB and geneD (sca4). In the present study the human pathogenic Rickettsia sp. strain Atlantic rainforest was identified in the state of Rio Grande do Sul, Southern Brazil. Since A. ovale, its main vector, is found frequently parasitizing dogs, animals that can cross international borders freely in southern Brazil, this bacteria can bring major concerns in terms of public health.

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

[EPIDEMIOLOGIC SITUATION BY NATURAL-FOCI INFECTIONS IN THE CRIMEA FEDERAL DISTRICT IN 2014-2015].

AIM: Analysis of epidemic manifestations of natural-foci infections (NFI), clarification of spectrum of their causative agents, determination of epizootic activity of natural foci in the Crimea Federal District (KFD). MATERIALS AND METHODS: Epizootologic examination of 10 administrative districts of KDF was carried out. 291 pools (2705 specimens) of ixodes ticks and 283 samples of organs of small mammals were studied by PCRmethod for the presence of DNA/RNA of causative agents of a number of NFI. RESULTS: Morbidity by NFI in KFD was registered by 6 nosologies: Lyme borreliosis, Marseilles fever, leptospirosis; tularemia, intestine yersiniosis and tick-borne viral encephalitis, wherein, transmissive infections made up 91.6%. Circulation of causative agents of Crimea hemorrhagic fever, Q fever, group of tick-borne spotted fever, Lyme borreliosis, human granulocytic anaplasmosis, human monocytic ehrlichiosis, hemorrhagic fever with renal syndrome, West Nile fever, tularemia and leptospirosis was established. CONCLUSION: Due to activity of natural foci of NFI further monitoring of epidemiologic and epizootologic manifestations of these infections in the Crimea, including using genetic methods of analysis, is necessary for ensuring sanitary-epidemiologic welfare of KFD population.

Host Responses and Regulation by NFκB Signaling in the Liver and Liver Epithelial Cells Infected with A Novel Tick-borne Bunyavirus.

Infection in humans by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus transmitted by ticks, is often associated with pronounced liver damage, especially in fatal cases. Little has been known, however, about how liver cells respond to SFTSV and how the response is regulated. In this study we report that proinflammatory cytokines were induced in liver tissues of C57/BL6 mice infected with SFTSV, which may cause tissue necrosis in mice. Human liver epithelial cells were susceptible to SFTSV and antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. We observed that infection of liver epithelial cells led to significant increases in proinflammatory cytokines and chemokines, including IL-6, RANTES, IP-10, and MIP-3a, which were regulated by NFκB signaling, and the activation of NFκB signaling during infection promoted viral replication in liver epithelial cells. Viral nonstructural protein NSs was inhibitory to the induction of IFN-ß, but interestingly, NFκB activation was enhanced in the presence of NSs. Therefore, NSs plays dual roles in the suppression of antiviral IFN-ß induction as well as the promotion of proinflammatory responses. Our findings provide the first evidence for elucidating host responses and regulation in liver epithelial cells infected by an emerging bunyavirus.

Characterization of spotted fever group Rickettsiae in ticks from a city park of Rome, Italy.

BACKGROUND: Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health. AIM AND METHODS: One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus, Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA. RESULTS: Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively. CONCLUSION: Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy.

Association of BoLA-DRB3 alleles with tick-borne disease tolerance in dairy cattle in a tropical environment.

Tick-borne disease is one of the most harmful tropical diseases in dairy production. Selection of dairy cows for tolerance to tick-borne disease is a challenging concept for dairy breeders in the tropics. The objectives of this study were (1) to detect specific tick-borne pathogen in cattle of different genetics and (2) to examine the polymorphisms of DRB3.2 alleles in Thai dairy cattle and find the allelic association with tick-borne disease tolerance. Specific primers to Anaplasma marginale (AM), Babesia bigemina (BG) and Babesia bovis (BB) were used to detect the infections by PCR. The results showed that the high proportion of infections were found in Bos indicus (Sahiwal, n=95) and crossbred Holstein × Zebu (75:25 Holstein:Zebu, n=101), compared to high Holstein fraction crossbreed (≥ 87.5% Holstein, n=187). The proportion of triple infections was also highly found in high Holstein fractions crossbreed. This study confirmed that Zebuine (Bos indicus) had a higher degree of tolerance, even when infected by tick-borne pathogens, compared to high Holstein fraction crossbred. The associated alleles of DRB3.2 for tick-borne pathogen infection tolerance were found: DRB3*14 and *41 were found to be tolerant to A. marginale; *14 to B. bovis; and *10 and *51 to B. bigemina. These tolerance alleles could be used as potential markers for selection in dairy genetic evaluation. The associated alleles for susceptibility were also found: *2 was found to be susceptible to A. marginale; *3 and *16 to B. bovis; and *20 to B. bigemina. These susceptibility alleles could be used as markers for culling, and selection favoring susceptibility alleles should be considered to maintain heterozygote advantage and pathogen-specific memories in the herd.

The RIPK2 gene: a positional candidate for tick burden supported by genetic associations in cattle and immunological response of knockout mouse.

Ticks and tick-borne diseases have a detrimental impact on livestock production causing estimated losses of around $200 million per year in Australia alone. Host resistance to ticks is heritable, within-breed heritability estimates being around 0.35, and with large differences between breeds. Previously a QTL for tick burden was detected on BTA14 at ~72 Mb distal to the centromere, near the gene receptor-interacting serine-threonine kinase 2 (RIPK2). To identify polymorphisms in this region, we sequenced all exons of the RIPK2 gene, identifying 46 single nucleotide polymorphism (SNP). Using SNP from RIPK2 as well as SNP from the bovine genome sequence, we genotyped two samples, one of 1,122 taurine dairy cattle and one of 761 zebu and zebu composite beef cattle. We confirmed that SNP and haplotypes from this region, including from RIPK2, were associated with tick burden in both dairy and beef cattle. To determine whether RIPK2 influences response to tick salivary gland extract (SGE), an immunisation experiment with tick SGE in a RIPK2 knockout (RIPK2 −/−) mouse strain was conducted. There was a significant (P < 0.05) reduction in IgG production in the RIPK2 −/− mouse in response to the SGE compared to its background strain C57BL/ 6 as well as the outbred CD1 mouse strain. In addition, antibodies generated by RIPK2 −/− mice recognised a different set of antigens within SGE when compared to parental-derived antibodies. In summary, the SNP association with tick burden at BTA14 was confirmed and quantitative and qualitative differences in antibody production were observed between RIPK2 −/− and wild-type mice.

Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens.

Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.

Total, membrane, and immunogenic proteomes of macrophage- and tick cell-derived Ehrlichia chaffeensis evaluated by liquid chromatography-tandem mass spectrometry and MALDI-TOF methods.

Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.