Tick Ornithodoros Research Articles

Detection of Rickettsia sp. strain Itinguçú in Ornithodoros faccinii (Acari: Argasidae) parasitizing the toad Rhinella ornata (Anura: Bufonidae) in Brazil

The pivotal role of amphibians in food webs and their value as indicators of disequilibrium in ecosystem health have long been recognized by wildlife biologists. However, massive pathogen-induced declines in global amphibian populations reported during the last 30 years served to alert the scientific community that knowledge of amphibian disease ecology, including parasitic and vector-borne conditions, was and remains incipient. Herein, we report the detection of a Rickettsia bacterium infecting larvae of the argasid tick Ornithodoros faccinii, collected from the toad Rhinella ornata, in Southeastern Brazil. Fragments of the genes 16S rDNA, gltA, htrA, sca1, sca4, and ompB were amplified by polymerase chain reaction (PCR), but the sequence encoding the ompA antigen was not detected. Nucleotide sequencing and multi-locus (gltA, htrA, sca1, and sca4) phylogenetic analyses characterized the bacterium, designated Rickettsia sp. strain Itinguçú, as a novel member of the spotted fever group (SFG) of the Rickettsia, closely related to the Rickettsia massiliae and to a lesser extent the Rickettsia helvetica subgroups. The apparent absence of the ompA protein together with limited levels of nucleotide (90.5 %) and amino acid (82–83 %) sequence identity, relative to the ompB gene of other species in the R. massiliae subgroup, were unusual features that may reflect adaptation to selective pressures exerted by the tick and/or amphibian immune systems. The ompB sequence was exploited to develop a low-cost method for differential identification of Rickettsia sp. strain Itinguçú, based on restriction fragment length polymorphism analysis of amplicons (PCR-RFLP). The characterization of this novel bacterium provided an unprecedented record of infection by an SFG Rickettsia in a member of the family Argasidae infesting a cold-blooded animal and raised the number of tick-associated Rickettsia reported in Brazil to sixteen. Moreover, it highlighted the value of and the requirement for continued and extended surveillance of wildlife as potential sources of emerging tick-borne pathogens.

Vector competence of the African argasid tick Ornithodoros moubata for the Q fever agent Coxiella burnetii.

Q fever is a widespread zoonotic disease caused by the intracellular bacterium Coxiella burnetii. While transmission is primarily but not exclusively airborne, ticks are usually thought to act as vectors on the basis of early microscopy studies. However, recent observations revealed that endosymbionts of ticks have been commonly misidentified as C. burnetii, calling the importance of tick-borne transmission into question. In this study, we re-evaluated the vector competence of the African soft tick Ornithodoros moubata for an avirulent strain of C. burnetii. To this end, we used an artificial feeding system to initiate infection of ticks, specific molecular tools to monitor further infections, and culture assays in axenic and cell media to check for the viability of C. burnetii excreted by ticks. We observed typical traits associated with vector competence: The exposure to an infected blood meal resulted in viable and persistent infections in ticks, trans-stadial transmissions of infection from nymphs to adults and the ability of adult ticks to transmit infectious C. burnetii. However, in contrast to early studies, we found that infection differed substantially between tick organs. In addition, while adult female ticks were infected, we did not observe C. burnetii in eggs, suggesting that transovarial transmission is not effective. Finally, we detected only a sporadic presence of C. burnetii DNA in tick faeces, but no living bacterium was further isolated in culture assays, suggesting that excretion in faeces is not a common mode of transmission in O. moubata.

Differential vector competence of Ornithodoros soft ticks for African swine fever virus: What if it involves more than just crossing organic barriers in ticks?

BackgroundSeveral species of soft ticks in genus Ornithodoros are known vectors and reservoirs of African swine fever virus (ASFV). However, the underlying mechanisms of vector competence for ASFV across Ornithodoros species remain to be fully understood. To that end, this study compared ASFV replication and dissemination as well as virus vertical transmission to descendants between Ornithodorosmoubata, O. erraticus, and O. verrucosus in relation to what is known about the ability of these soft tick species to transmit ASFV to pigs. To mimic the natural situation, a more realistic model was used where soft ticks were exposed to ASFV by allowing them to engorge on viremic pigs.MethodsOrnithodoros moubata ticks were infected with the ASFV strains Liv13/33 (genotype I) or Georgia2007/1 (genotype II), O. erraticus with OurT88/1 (genotype I) or Georgia2007/1 (genotype II), and O. verrucosus with Ukr12/Zapo (genotype II), resulting in five different tick–virus pairs. Quantitative PCR (qPCR) assays targeting the VP72 ASFV gene was carried out over several months on crushed ticks to study viral replication kinetics. Viral titration assays were also carried out on crushed ticks 2 months post infection to confirm virus survival in soft ticks. Ticks were dissected. and DNA was individually extracted from the following organs to study ASFV dissemination: intestine, salivary glands, and reproductive organs. DNA extracts from each organ were tested by qPCR. Lastly, larval or first nymph-stage progeny emerging from hatching eggs were tested by qPCR to assess ASFV vertical transmission.ResultsComparative analyses revealed higher rates of ASFV replication and dissemination in O. moubata infected with Liv13/33, while the opposite was observed for O. erraticus infected with Georgia2007/1 and for O. verrucosus with Ukr12/Zapo. Intermediate profiles were found for O. moubata infected with Georgia2007/1 and for O. erraticus with OurT88/1. Vertical transmission occurred efficiently in O. moubata infected with Liv13/33, and at very low rates in O. erraticus infected with OurT88/1.ConclusionsThis study provides molecular data indicating that viral replication and dissemination in Ornithodoros ticks are major mechanisms underlying ASFV horizontal and vertical transmission. However, our results indicate that other determinants beyond viral replication also influence ASFV vector competence. Further research is required to fully understand this process in soft ticks.Graphical

A comparison of the cuticular properties of argasid and ixodid ticks: Ornithodoros moubata (Argasidae) vs. Amblyomma hebraeum and Ixodes pacificus (Ixodidae)

Ticks (Chelicerata, Ixodida) are blood-feeding ectoparasites believed to have evolved at least about 120 millions of years ago and found worldwide. However, many aspects of their unique life cycle and anatomy, including their mechanical properties, remain to be understood. Here, we compared the mechanical properties of the cuticle of the argasid tick Ornithodoros moubata to those of two species of ixodid tick, Amblyomma hebraeum and Ixodes pacificus that we explored in our earlier studies of the tick exoskeleton. Significant differences were expected given the substantial difference in life cycle, including a five-fold increase during the repeated adult blood meal for female O. moubata vs. 70- to 120-fold during the single feeding of the adult female A. hebraeum and I. pacificus. We demonstrate here that the layered structure and mechanical properties (stiffness and viscosity) of the cuticle show minor differences, but the difference in cuticle thickness is substantial. Ductility is lost during feeding; reduced pH restores ductility. Previous work suggests that this occurs in vivo in engorged ixodid ticks; there is no evidence of this occurring in vivo in O. moubata. Thinning of cuticle in O. moubata fed females is consistent with the predicted stretch of cuticle due to the blood meal; there is no evidence of cuticle synthesis during the short feeding period. Dimensional analysis suggests that the soft feel of argasid ticks is related to cuticle thickness, not cuticle stiffness. Relative to argasid ticks, the hard ixodid ticks accommodate a ca. 20-fold higher size of blood meal by starting with a thicker cuticle and growing much additional cuticle during engorgement.

Evaluating Functional Dispersal in a Nest Ectoparasite and Its Eco-Epidemiological Implications.

Functional dispersal (between-site movement, with or without subsequent reproduction) is a key trait acting on the ecological and evolutionary trajectories of a species, with potential cascading effects on other members of the local community. It is often difficult to quantify, and particularly so for small organisms such as parasites. Understanding this life history trait can help us identify the drivers of population dynamics and, in the case of vectors, the circulation of associated infectious agents. In the present study, functional dispersal of the soft tick Ornithodoros maritimus was studied at a small scale, within a colony of yellow-legged gulls (Larus michahellis). Previous work showed a random distribution of infectious agents in this tick at the within-colony scale, suggesting frequent tick movement among nests. This observation contrasts with the presumed strong endophilic nature described for this tick group. By combining an experimental field study, where both nest success and tick origin were manipulated, with Capture-Mark-Recapture modeling, dispersal rates between nests were estimated taking into account tick capture probability and survival, and considering an effect of tick sex. As expected, tick survival probability was higher in successful nests, where hosts were readily available for the blood meal, than in unsuccessful nests, but capture probability was lower. Dispersal was low overall, regardless of nest state or tick sex, and there was no evidence for tick homing behavior; ticks from foreign nests did not disperse more than ticks in their nest of origin. These results confirm the strong endophilic nature of this tick species, highlighting the importance of life cycle plasticity for adjusting to changes in host availability. However, results also raise questions with respect to the previously described within-colony distribution of infectious agents in ticks, suggesting that tick dispersal either occurs over longer temporal scales and/or that transient host movements outside the breeding period result in vector exposure to a diverse range of infectious agents.

The impact of in vitro cultivation on the natural life cycle of the tick-borne relapsing fever spirochete Borrelia turicatae.

Tick-borne relapsing fever is an infectious disease caused by Borrelia species and are primarily transmitted by Ornithodoros ticks. Prior work indicated that in vitro cultivated spirochetes remain infectious to mice by needle inoculation; however, the impact of laboratory propagation on the pathogens natural life cycle has not been determined. Our current study assessed the effect of serial cultivation on the natural tick-mammalian transmission cycle. First, we evaluated genomic DNA profiles from B. turicatae grown to 30, 60, 120, and 300 generations, and these spirochetes were used to needle inoculate mice. Uninfected nymphal ticks were fed on these mice and acquisition, transstadial maintenance, and subsequent transmission after tick bite was determined. Infection frequencies in mice that were fed upon by ticks colonized with B. turicatae grown to 30, 60, and 120 generations were 100%, 100%, and 30%, respectively. Successful infection of mice by tick feeding was not detected after 120 generations. Quantifying B. turicatae in tick tissues indicated that by 300 generations they no longer colonized the vector. The results indicate that in vitro cultivation significantly affects the establishment of tick colonization and murine infection. This work provides a foundation for the identification of essential genetic elements in the tick-mammalian infectious cycle.

Identification of African swine fever virus-like elements in the soft tick genome provides insights into the virus\u2019 evolution

BackgroundAfrican swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus’ evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host’s genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery.ResultsFor the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks.ConclusionHere, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector ‘arms race’, a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.

Changes in Bacterial Diversity, Composition and Interactions During the Development of the Seabird Tick Ornithodoros maritimus (Argasidae).

Characterising within-host microbial interactions is essential to understand the drivers that shape these interactions and their consequences for host ecology and evolution. Here, we examined the bacterial microbiota hosted by the seabird soft tick Ornithodoros maritimus (Argasidae) in order to uncover bacterial interactions within ticks and how these interactions change over tick development. Bacterial communities were characterised through next-generation sequencing of the V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene. Bacterial co-occurrence and co-exclusion were determined by analysing networks generated from the metagenomic data obtained at each life stage. Overall, the microbiota of O. maritimus was dominated by four bacterial genera, namely Coxiella, Rickettsia, Brevibacterium and Arsenophonus, representing almost 60% of the reads. Bacterial diversity increased over tick development, and adult male ticks showed higher diversity than did adult female ticks. Bacterial networks showed that co-occurrence was more frequent than co-exclusion and highlighted substantial shifts across tick life stages; interaction networks changed from one stage to the next with a steady increase in the number of interactions through development. Although many bacterial interactions appeared unstable across life stages, some were maintained throughout development and were found in both sexes, such as Coxiella and Arsenophonus. Our data support the existence of a few stable interactions in O. maritimus ticks, on top of which bacterial taxa accumulate from hosts and/or the environment during development. We propose that stable associations delineate core microbial interactions, which are likely to be responsible for key biological functions.

General Microbiota of the Soft Tick Ornithodoros turicata Parasitizing the Bolson Tortoise (Gopherus flavomarginatus) in the Mapimi Biosphere Reserve, Mexico.

The general bacterial microbiota of the soft tick Ornithodoros turicata found on Bolson tortoises (Gopherus flavomarginatus) were analyzed using next generation sequencing. The main aims of the study were to establish the relative abundance of bacterial taxa in the tick, and to document the presence of potentially pathogenic species for this tortoise, other animals, and humans. The study was carried-out in the Mapimi Biosphere Reserve in the northern-arid part of Mexico. Bolson tortoises (n = 45) were inspected for the presence of soft ticks, from which 11 tortoises (24.4%) had ticks in low loads (1–3 ticks per individual). Tick pools (five adult ticks each) were analyzed through 16S rRNA V3–V4 region amplification in a MiSeq Illumina, using EzBioCloud as a taxonomical reference. The operational taxonomic units (OTUs) revealed 28 phyla, 84 classes, 165 orders, 342 families, 1013 genera, and 1326 species. The high number of taxa registered for O. turicata may be the result of the variety of hosts that this tick parasitizes as they live inside G. flavomarginatus burrows. While the most abundant phyla were Proteobacteria, Actinobacteria, and Firmicutes, the most abundant species were two endosymbionts of ticks (Midichloria-like and Coxiella-like). Two bacteria documented as pathogenic to Gopherus spp. were registered (Mycoplasma spp. and Pasteurella testudinis). The bovine and ovine tick-borne pathogens A. marginale and A. ovis, respectively, were recorded, as well as the zoonotic bacteria A. phagocytophilum, Coxiella burnetii, and Neoehrlichia sp. Tortoises parasitized with O. turicata did not show evident signs of disease, which could indicate a possible ecological role as a reservoir that has yet to be demonstrated. In fact, the defense mechanisms of this tortoise against the microorganisms transmitted by ticks during their feeding process are still unknown. Future studies on soft ticks should expand our knowledge about what components of the microbiota are notable across multiple host–microbe dynamics. Likewise, studies are required to better understand the host competence of this tortoise, considered the largest terrestrial reptile in North America distributed throughout the Chihuahuan Desert since the late Pleistocene.

Detection of African swine fever virus genotype XV in a sylvatic cycle in Saadani National Park, Tanzania.

African swine fever (ASF) is a severe haemorrhagic disease of domestic pigs caused by ASF virus (ASFV). ASFV is transmitted by soft ticks (Ornithodoros moubata complex group) and by direct transmission. In Africa, ASF is maintained in transmission cycles of asymptomatic infection involving wild suids, mainly warthogs (Phacochoerus africanus). ASF outbreaks have been reported in many parts of Tanzania; however, active surveillance has been limited to pig farms in a few geographical locations. There is an information gap on whether and where the sylvatic cycle may occur independently of domestic pigs. To explore the existence of a sylvatic cycle in Saadani National Park in Tanzania, blood and serum samples were collected from 19 warthogs selected using convenience sampling along vehicle‐accessible transects within the national park. The ticks were sampled from warthog burrows. Blood samples and ticks were subjected to ASFV molecular diagnosis (PCR) and genotyping, and warthog sera were subjected to serological (indirect ELISA) testing for ASFV antibody detection. All warthog blood samples were PCR‐negative, but 16/19 (84%) of the warthog sera were seropositive by ELISA confirming exposure of warthogs to ASFV. Of the ticks sampled, 20/111 (18%) were positive for ASFV by conventional PCR. Sequencing of the p72 virus gene fragments showed that ASF viruses detected in ticks belonged to genotype XV. The results confirm the existence of a sylvatic cycle of ASFV in Saadani National Park, Tanzania, that involves ticks and warthogs independent of domestic pigs. Our findings suggest that genotype XV previously reported in 2008 in Tanzania is likely to be widely distributed and involved in both wild and domestic infection cycles. Whole‐genome sequencing and analysis of the ASFV genotype XV circulating in Tanzania is recommended to determine the phylogeny of the viruses.

A new piroplasmid species infecting dogs: morphological and molecular characterization and pathogeny of Babesia negevi n. sp.

IntroductionBabesiosis is a protozoan tick-borne infection associated with anemia and life-threatening disease in humans, domestic and wildlife animals. Dogs are infected by at least six well-characterized Babesia spp. that cause clinical disease. Infection with a piroplasmid species was detected by light microscopy of stained blood smears from five sick dogs from Israel and prompted an investigation on the parasite’s identity.MethodsGenetic characterization of the piroplasmid was performed by PCR amplification of the 18S rRNA and the cytochrome c oxidase subunit 1 (cox1) genes, DNA sequencing and phylogenetic analysis. Four of the dogs were co-infected with Borrelia persica (Dschunkowsky, 1913), a relapsing fever spirochete transmitted by the argasid tick Ornithodoros tholozani Laboulbène & Mégnin. Co-infection of dogs with B. persica raised the possibility of transmission by O. tholozani and therefore, a piroplasmid PCR survey of ticks from this species was performed.ResultsThe infected dogs presented with fever (4/5), anemia, thrombocytopenia (4/5) and icterus (3/5). Comparison of the 18S rRNA and cox1 piroplasmid gene sequences revealed 99–100% identity between sequences amplified from different dogs and ticks. Phylogenetic trees demonstrated a previously undescribed species of Babesia belonging to the western group of Babesia (sensu lato) and closely related to the human pathogen Babesia duncani Conrad, Kjemtrup, Carreno, Thomford, Wainwright, Eberhard, Quick, Telfrom & Herwalt, 2006 while more moderately related to Babesia conradae Kjemtrup, Wainwright, Miller, Penzhorn & Carreno, 2006 which infects dogs. The piroplasm forms detected included tetrads (Maltese cross), merozoite and trophozoite stages whose average size was larger than stages of other canine Babesia spp. belonging to the Babesia (s.l.) and B. gibsoni Patton, 1910, and smaller than other canine Babesia (sensu stricto) spp. Of 212 O. tholozani ticks surveyed, 11 (5.2%) harbored DNA of the new species of Babesia.ConclusionsBabesia negevi n. sp. is described based on morphological and genetic characterization and phylogenetic analyses. The species is named after the Negev desert of southern Israel, where the first infected dog originated from. Despite co-infection in four dogs, the fifth dog had fatal disease attesting that B. negevi n. sp. infection requires clinical attention. Incriminating O. tholozani or another tick species as the vector of Babesia negevi n. sp., would require additional studies.

Successful Infection of Domestic Pigs by Ingestion of the European Soft Tick O. Erraticus That Fed on African Swine Fever Virus Infected Pig.

African swine fever is a highly lethal hemorrhagic fever of Suidae, threatening pig production globally. Suidae can be infected by different ways like ingestion of contaminated feed, direct contact with infected animals or fomites, and biting by infected soft tick bites. As already described, European soft ticks (Ornithodoros erraticus and Ornithodoros verrucosus) were not able to transmit African swine fever virus by biting pigs although these ticks maintained the infectious virus during several months; therefore, the possibility for pigs to become infected through the ingestion of infected ticks was questioned but not already explored. To determine if such oral ingestion is an alternative pathway of transmission, O. erraticus ticks were infected by blood-feeding on a viremic pig infected with the European African swine fever virus strain Georgia2007/1, then frozen at zero and two months post-engorgement, then after, were embedded in the food to pigs. Pig infection was successful, with superior efficiency with ticks frozen just after the infectious blood meal. These results confirmed the potential role of O. erraticus ticks as an ASFV reservoir and demonstrated the efficiency of non-conventional pathways of transmission.

Redescription of the larva, and description of the nymphal and adult stages of Ornithodoros peruvianus Kohls, Clifford & Jones, 1969 (Acari: Argasidae).

The soft tick Ornithodoros peruvianus Kohls, Clifford & Jones, 1969 was described as a parasite of bats in Peru upon the examination of engorged larvae only. Recently, larvae of this tick species were reported on bats from northern Chile. However, the adult and nymphal stages of O. peruvianus have remained undescribed. This study aimed to redescribe the larva of O. peruvianus based on unfed specimens, and to describe nymphs, the male and the female of this species. Ticks were collected on the walls inside three caves in northern Chile. Two females laid eggs in the laboratory. Part of the unfed larvae was separated for morphological and morphometrical analyses, and the remaining specimens were fed upon laboratory mice in order to obtain subsequent nymphal and adult stages. The first nymphal stage (N1) moulted either to male or to a second nymphal stage (N2) without feeding. Obtained N2 moulted either to male or female after one meal. PCR amplification of tick mitochondrial 16S rRNA of specimens from the three caves revealed almost identical sequences. The unfed larva of O. peruvianus has an elongated idiosoma, and fringed setae cover the ventral surfaces of coxae, palps and tarsi. Nymph 1 has a thin integument covered by incipient mammillae and barely noticeable dorsal disks; it lacks cheeks and possesses few short setae on the basis capitulum. Nymph 2 has a pair of small cheeks and resembles adult stages in its tegumentary traits and capitulum. Adult stages exhibit developed cheeks (larger in females) without the capacity to completely cover the capitulum. Very small and low mammillae cover the surface of the dorsal idiosoma in adults. As this feature also occurs in other bat-associated soft ticks, regardless of their phylogenetic relatedness, small mammillae in bat soft ticks are suggestive of convergent evolution.

African swine fever: A permanent threat to Indian pigs.

:India has 9 million pigs, of which 45% are in the North eastern (NE) states of India. Viral diseases affecting pigs are a major concern of mortality causing huge loss to the pig farmers. One such disease is African swine fever (ASF) that has already knocked the porous borders of NE states of India. ASF is a highly contagious devastating disease of pigs and wild boars causing 100% mortality. The causative agent African swine fever virus (ASFV) belongs to the genus Asfivirus, family Asfarviridae. Pig is the only species affected by this virus. Soft ticks (Ornithodoros genus) are shown to be reservoir and transmission vectors of ASFV. Transmission is very rapid and quickly engulfs the entire pig population. It is very difficult to differentiate classical swine fever from ASF since clinical symptoms overlap. Infected and in contact pigs should be culled immediately and buried deep, and sheds and premises be disinfected to control the disease. There is no vaccine available commercially. Since its first report in Kenya in 1921, the disease has been reported from the countries in Europe, Russian federation, China, and Myanmar. The disease is a threat to Indian pigs. OIE published the first report of ASF in India on May 21, 2020, wherein, a total of 3701 pigs died from 11 outbreaks (Morbidity - 38.45% and mortality - 33.89%) in Assam and Arunachal Pradesh states of India. ASF is non-zoonotic.

Efficacy and safety of ethanolic Curcuma longa extract as a treatment for sand tampan ticks in a rabbit model.

Background and Aim:The soft tick Ornithodoros savignyi is distributed throughout Africa, including Egypt. It primarily attacks camels, cattle, donkeys, and cows; and rarely affects humans. This study evaluated the acaricidal efficacy of ethanolic Curcuma longa extract (Turmeric) on the second nymphs of O. savignyi and then investigated the safety of this herb in rabbits.Materials and Methods:The nymphs were immersed in 10, 5, 2.5, 1.25, and 0.625 mg/ml ethanolic C. longa extract. An additional group was immersed in ethanol as a control. On the 1st, 7th, and 15th-day post-treatment, the mortality percentages, LC50, and LC95 were calculated. The ticks exposed to 10mg/ml ethanol C. longa extract were investigated by scanning electron microscopy (SEM). Three male New Zealand White rabbits were orally administered 2ml (two doses) of 10mg/ml ethanolic C. longa extract, and another three rabbits were orally given two doses of 2ml of absolute ethanol as a negative control. Histopathological examination of the kidney and liver hematology and the kidney and liver function was performed. Chemical analysis of the extract was determined by gas chromatography-mass spectrometry (GC-MS).Results:The LC50 and LC95 were 1.31 and 15.07, 1.07 and 8.56, and 0.81 and 6.97mg/ml on the 1st, 7th, and 15thday, respectively. SEM revealed that mamillae and spots on the surfaces of the treated ticks were not discriminating except for some clefts on the surfaces. The histological examination, blood profile, and biochemical analyses revealed no significant differences between the treated and untreated rabbits (p>0.05). GC/MS analysis revealed 50 compounds, and curcumene and tumerone were found to be the major constituents of this ethanolic extract.Conclusion:The ethanolic C. longa extract produced a strong acaricidal effect on the second nymph of O. savignyi, and it was safe to use in rabbits.

A soft tick Ornithodoros moubata salivary protein OmCI is a potent inhibitor to prevent avian complement activation

Complement is a key first line innate host defense system in the blood of vertebrates. Upon activation, this powerful defense mechanism can elicit inflammatory responses, lyse non-self-cells, or mark them for opsonophagocytic removal. Blood-feeding arthropods thus require the ability to block host complement activation in the bloodmeal to prevent undesired cell or tissue damage during feeding. The soft tick Ornithodoros moubata produces a complement inhibitory protein, OmCI. This protein binds to a mammalian complement protein C5 and blocks further activation of complement cascades, which results in the prevention of complement-mediated bacterial killing through membrane attack complex. Interestingly, the amino acids involved in OmCI binding are highly conserved among mammalian and avian C5, but the ability of this protein to inhibit the complement from birds remains unclear. Here we demonstrated that OmCI is capable of preventing quail complement-mediated erythrocyte lysis, inhibiting the capability of this animal’s complement to eliminate a serum-sensitive Lyme disease bacterial strain. We also found that the ability of OmCI to inhibit quail complement-mediated killing of Lyme disease bacteria can be extended to different domestic and wild birds. Our results illustrate the utility of OmCI to block bird complement. These results provide the foundation for further use of this protein as a tool to study the molecular basis of avian complement and pathogen evasion to such a defense mechanism.

Karyotype changes in cultivated tick cell lines

Background. Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and different aspects of tick physiology. However, long-term cultivation of tick cells can influence genome stability. The aim of our work was to analyze and compare the karyotypes of Ixodes scapularis, I. ricinus, and Ornithodoros moubata tick cell lines after long-term been in culture. Methods. Mitotic spreads were prepared to count the number of chromosomes in ISE6, ISE18, IRE11, IRE/CTVM19, IRE/CTVM20, and OME/CTVM22 cell lines. The genome size of tick cell cultures was estimated by flow cytometry using propidium iodide staining. Results. The modal chromosome numbers around 22 and around 48 were typical for both I. ricinus and I. scapularis cell lines and differed from the diploid chromosome number in Ixodesticks – 28. In the OME/CTVM22cell line, the modal chromosome number was 33, instead of 20 in Ornithodoros ticks. All tick cell lines had a larger genome size in compare to genomes of parental ticks. Conclusions.Tick cell lines can be used for research purposes, however, differences in the internal processes between different cell populations should be taken into account. Acknowledgments: We thank Lesley Bell-Sakyi and the Tick Cell Biobank for provision of the tick cell lines.

Antimicrobial function of short amidated peptide fragments from the tick-derived OsDef2 defensin.

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3μM against Escherichia coli. Killing was reached within 10minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.

In silico selection of functionally important proteins from the mialome of Ornithodoros erraticus ticks and assessment of their protective efficacy as vaccine targets

BackgroundNew candidate protective antigens for tick vaccine development may be identified by selecting and testing antigen candidates that play key biological functions. After blood-feeding, tick midgut overexpresses proteins that play essential functions in tick survival and disease transmission. Herein, Ornithodoros erraticus midgut transcriptomic and proteomic data were examined in order to select functionally significant antigens upregulated after feeding to be tested as vaccine candidate antigens.MethodsTranscripts annotated as chitinases, tetraspanins, ribosomal protein P0 and secreted proteins/peptides were mined from the recently published O. erraticus midgut transcriptome and filtered in a second selection step using criteria based on upregulation after feeding, predicted antigenicity and expression in the midgut proteome. Five theoretical candidate antigens were selected, obtained as recombinant proteins and used to immunise rabbits: one chitinase (CHI), two tetraspanins (TSPs), the ribosomal protein P0 (RPP0) and one secreted protein PK-4 (PK4).ResultsRabbit vaccination with individual recombinant candidates induced strong humoral responses that mainly reduced nymph moulting and female reproduction, providing 30.2% (CHI), 56% (TSPs), 57.5% (RPP0) and 57.8% (PK4) protection to O. erraticus infestations and 19.6% (CHI), 11.1% (TSPs), 0% (RPP0) and 8.1% (PK4) cross-protection to infestations by the African tick Ornithodoros moubata. The joint vaccine efficacy of the candidates was assessed in a second vaccine trial reaching 66.3% protection to O. erraticus and 25.6% cross-protection to O. moubata.ConclusionsThese results (i) indicate that argasid chitinases and RPP0 are promising protective antigens, as has already been demonstrated for ixodid chitinases and RPP0, and could be included in vaccines targeting multiple tick species; (ii) reveal novel protective antigens tetraspanins and secreted protein PK-4, never tested before as protective antigens in ticks; and (iii) demonstrate that multi-antigenic vaccines increased vaccine efficacy compared with individual antigens. Lastly, our data emphasize the value of the tick midgut as a source of protective candidate antigens in argasids for tick control.

Antibodies to Borrelia turicatae in Experimentally Infected Dogs Cross-React with Borrelia burgdorferi Serologic Assays.

Tick-borne relapsing fever (TBRF) is caused by several Borrelia spp. (including Borrelia turicatae), which are primarily transmitted by Ornithodoros ticks. Relapsing fever group species are found worldwide, except for Antarctica. Approximately 500 human cases were reported between 1990 and 2011 in the United States (likely an underestimate), while cases in domestic and wild dogs were reported from Florida, Texas, and Washington. TBRF spirochetes are related to Borrelia burgdorferi, the agent of Lyme borreliosis. Dogs are routinely screened for B. burgdorferi, but it is unknown if infection with TBRF agents produces antibodies cross-reactive with B. burgdorferi assays. These data are critical for accurate surveillance of TBRF and Lyme borreliosis in dogs. In this study, B. burgdorferi-negative dogs were inoculated with B. turicatae, and seroconversion was confirmed by the rBipA (recombinant Borrelia immunogenic protein A) Western blot. Seropositive samples were tested with commercial and veterinary diagnostic laboratory B. burgdorferi-based tests. Borrelia turicatae-seroreactive samples cross-reacted with a whole-cell indirect fluorescent antibody (IFA) test and two multiantigen tests, but not with single-antigen tests using C6. Cross-reactivity with TBRF can confound epidemiology and surveillance efforts and confuse recommendations made by veterinarians for prevention and control. These findings demonstrate the need to critically evaluate results from B. burgdorferi diagnostic tests in the context of the assay type and the animal's geographical location and history of travel, as well as highlighting the need for commercially available specific diagnostic tests for TBRF spirochetes.