Background: Host choice and feeding success of sand fly vectors of visceral leishmaniasis (VL) are important factors in understanding the epidemiology and for developing efficient control strategies. The aim of the present study was to determine the host preference of Phlebotomus orientalis in the VL focus of Tahtay Adiyabo district, northern Ethiopia. Methods: Two separate experiments were conducted testing attraction to humans, domestic animals, and small wild animals. The host choice of P. orientalis and other sand fly species was assessed using tent traps baited with seven different animals (human, cow, sheep, goat, donkey, dog and chicken) and a blank control. Baited traps were rotated every night in a Latin square design for two consecutive full rounds totaling 16 trap-nights. The second set of experiments tested attraction to small wild animals including; ground squirrel (Xerus rutilus), hare (Lepus sp.), gerbil (Tatera robusta) and spiny mouse (Acomys cahirinus). Animals were caged in standard rodent traps or cylindrical wire-mesh cages. The bait animals were placed in agricultural field and the attracted sand flies were collected using unlit CDC traps for 10 trapping nights. Sand fly specimens collected from each of the experiments were identified to species level and counted. Results: Significant difference (P < 0.05) was observed in the attraction and feeding rate of P. orientalis to different baits. In the first experiment, cow-baited tent traps attracted the highest mean number of P. orientalis (mean = 510 flies). The engorgement rate of P. orientalis on donkey was the highest followed by cow, and much lower on goat, sheep, dog and chicken. In the case of smaller wild animals, more numbers of P. orientalis females were attracted to squirrels followed by hares, gerbils and the spiny rat. However, the engorgement rates for P. orientalis in the smaller animals were very low (1.08%) compared with larger domestic animals (30.53%). Conclusion: The tendency of female P. orientalis to engorge in large numbers on certain species of domestic as well as wild animals strongly indicated that the species is primarily zoophilic in its host preference with feeding habits that may vary depending on the availability of hosts.

Background: The low prevalence of blood parasites in some bird species may be related to the habitats they frequent, the inexistence of the right host-parasite assemblage or the immunological capacity of the host. Here, we assess the parasite load of breeding populations of Eleonora’s falcon (Falco eleonorae), a medium-sized long-distance migratory raptor that breeds on small isolated islets throughout the Mediterranean basin and overwinters in inland Madagascar. Methods: We examined the prevalence and genetic diversity of the blood parasites belonging to the genera Plasmodium, Haemoproteus and Leucocytozoon in Eleonora’s falcon nestlings from five colonies and in adults from two colonies from nesting sites distributed throughout most of the species’ breeding range. Results: None of the 282 nestlings analysed were infected by blood parasites; on the other hand, the lineages of Plasmodium, Haemoproteus and Leucocytozoon were all found to infect adults. Our results support the idea of no local transmission of vector-borne parasites in marine habitats. Adult Eleonora’s falcons thus may be infected by parasites when on migration or in their wintering areas. Conclusion: The characteristics of marine environments with a lack of appropriate vectors may thus be the key factor determining the absence of local transmission of blood parasites. By comparing the parasite lineages isolated in this species with those previously found in other birds we were able to infer the most likely areas for the transmission of the various parasite lineages.

by Pontus Lindblom, Peter Wilhelmsson, Linda Fryland, Andreas Matussek, Mats Haglund, Johanna Sjöwall, Sirkka Vene, Dag Nyman, Pia Forsberg, Per-Eric Lindgren

We performed a cross-sectional study including 533 individuals (median age 61) from the highly TBE endemic Åland Islands in the archipelago between Sweden and Finland. Blood samples, questionnaires and vaccination records were obtained from all study participants. The aim was to investigate if there was any association between TBEV antibody titer and 12 health-related factors. Measurement of TBEV IgG antibodies was performed using two commercial ELISA assays (Enzygnost and Immunozym), and a third in-house rapid fluorescent focus inhibition test was used to measure TBEV neutralizing antibodies. The age of the individual and the number of vaccine doses were the two most important factors determining the immunological response to vaccination. The response to each vaccine dose declined linearly with increased age. A 35 year age difference corresponds to a vaccine dose increment from 3 to 4 to achieve the same immunological response. Participants previously vaccinated against other flaviviruses had lower odds of being seropositive for neutralizing TBEV antibodies on average, while participants with self-reported asthma had higher odds of being seropositive. By comparing the 3 serological assays we show that the Enzygnost and Immunozym assay differ due to choice of cutoffs, but not in overall accuracy.

by Sarah Bonnet, Lorraine Michelet, Sara Moutailler, Justine Cheval, Charles Hébert, Muriel Vayssier-Taussat, Marc Eloit

Background

Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.

Methodology/Principal Findings

We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.

Conclusions/Significance

Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

by Attila D. Sándor, Daniel I. Mărcuţan, Gianluca D'Amico, Călin M. Gherman, Mirabela O. Dumitrache, Andrei D. Mihalca

Migratory birds play important roles as distributors of ticks within and between continents. In the Old World, the most important migratory route of birds links Asia, Europe and Africa. During their migration, birds use various stopover sites, where they feed and rest and where ticks may attach or detach, creating new natural foci for vector-borne diseases. Danube Delta is one of the most important migration hotspots and so far no studies were focused on ticks of migratory birds herein. The aim of the present study was to assess the species diversity and seasonal dynamics of ticks parasitizing migratory birds in Danube Delta Biosphere Reserve. Migratory birds were trapped on Grindul Lupilor (44°41′N; 28°56′E) using mist nets during 4 migratory seasons (2 spring and 2 autumn) in 2011 and 2012. From each bird, all the ticks were collected and identified based on morphological features. Epidemiological parameters (prevalence, mean abundance, mean intensity) were calculated and all data were analysed statistically based on the season (spring and autumn), regional status of birds (migrants and breeding) and foraging behaviour (ground feeders, reed-bed feeders, foliage feeders). A total of 1434 birds (46 species) were captured. Ticks were found on 94 birds (10 species). Significantly more migratory birds hosted ticks, compared to resident birds. The 400 collected ticks belonged to four species: Ixodes ricinus (92.25%), I. arboricola (6.25%), I. redikorzevi (1.00%) and Haemaphysalis punctata (0.50%). A higher prevalence was found for I. ricinus in spring, with higher prevalence of nymphs in this season, while larvae occurred with the same prevalence in both seasons. Larval intensity was higher during spring and nymphs were more abundant during autumn. The seasonal differences in our study may be related not to the local seasonal dynamics of ticks, but on the seasonal dynamics at the site of migration initiation.

by Fauziah Mohd Jaafar, Mourad Belhouchet, Manjunatha Belaganahalli, Robert B. Tesh, Peter P. C. Mertens, Houssam Attoui

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein ‘outer-capsid protein 1′ (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 ‘subcore-shell protein’ as the third largest protein ‘VP3(T2)’ in the first of these groups, but the second largest protein ‘VP3(T2)’ in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors.

by Muriel Vayssier-Taussat, Sara Moutailler, Lorraine Michelet, Elodie Devillers, Sarah Bonnet, Justine Cheval, Charles Hébert, Marc Eloit

Background and Aims

Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France.

Methods

RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence.

Results

We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas.

Conclusions

The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.

by Andrea Swei, Brandy J. Russell, Samia N. Naccache, Beniwende Kabre, Narayanan Veeraraghavan, Mark A. Pilgard, Barbara J. B. Johnson, Charles Y. Chiu

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.

by Natalia S. Pripuzova, Larissa V. Gmyl, Lidiya Iu. Romanova, Natalia V. Tereshkina, Yulia V. Rogova, Liubov L. Terekhina, Liubov I. Kozlovskaya, Mikhail F. Vorovitch, Karina G. Grishina, Andrey V. Timofeev, Galina G. Karganova

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.