Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation.

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.

Failed Disruption of Tick Feeding, Viability, and Molting after Immunization of Mice and Sheep with Recombinant Ixodes ricinus Salivary Proteins IrSPI and IrLip1.

To identify potential vaccine candidates against Ixodes ricinus and tick-borne pathogen transmission, we have previously sequenced the salivary gland transcriptomes of female ticks infected or not with Bartonella henselae. The hypothesized potential of both IrSPI (I. ricinus serine protease inhibitor) and IrLip1 (I. ricinus lipocalin 1) as protective antigens decreasing tick feeding and/or the transmission of tick-borne pathogens was based on their presumed involvement in dampening the host immune response to tick feeding. Vaccine endpoints included tick larval and nymphal mortality, feeding, and molting in mice and sheep. Whether the antigens were administered individually or in combination, the vaccination of mice or sheep elicited a potent antigen-specific antibody response. However, and contrary to our expectations, vaccination failed to afford protection against the infestation of mice and sheep by I. ricinus nymphs and larvae, respectively. Rather, vaccination with IrSPI and IrLip1 appeared to enhance tick engorgement and molting and decrease tick mortality. To the best of our knowledge, these observations represent the first report of induction of vaccine-mediated enhancement in relation to anti-tick vaccination.

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

First identification of Rickettsia helvetica in questing ticks from a French Northern Brittany Forest.

Tick-borne rickettsiae are considered to be emerging, but data about their presence in western Europe are scarce. Ixodes ricinus ticks, the most abundant and widespread tick species in western Europe, were collected and tested for the presence of several tick-borne pathogens in western France, a region never previously explored in this context. There was a high tick abundance with a mean of 4 females, 4.5 males, and 23.3 nymphs collected per hour per collector. Out of 622 tested ticks, specific PCR amplification showed the presence of tick symbionts as well as low prevalence of Borrelia burgdorferi (0.8%), Bartonella spp. (0.17%), and Anaplasma phagocytophilum (0.09%). The most prevalent pathogen was Rickettsia helvetica (4.17%). This is the first time that this bacteria has been detected in ticks in this region, and this result raises the possibility that bacteria other than those classically implicated may be involved in rickettsial diseases in western France.

Environmental factors influencing tick densities over seven years in a French suburban forest.

BACKGROUND: Worldwide changes in socio-economic and environmental factors and the global climate are recognised causes of variation in tick distribution and density. Thus it is of great importance that new studies address the changing risk of infection for exposed populations. In Europe, Ixodes ricinus ticks are the most common vectors of several pathogens impacting veterinary and public health that have colonised suburban habitats. METHODS: This study aimed to evaluate longitudinal I. ricinus questing densities and infection rates over 7 years in a French suburban forested area with high human population density. Ticks were collected in spring yearly between 2008 and 2014 and, out of a total of 8594 collected I. ricinus, a representative subset of adult females (n = 259) were individually examined for the presence of several pathogens via PCR. RESULTS: Nymph densities peaked in 2009-2011, and then declined in 2012-2014. Changes in monthly temperature only had a modest impact on this variation. In contrast, analysis revealed a complex intra-annual relationship between mean nymph density and both concurrent and lagged mean monthly temperatures. The following pathogens were detected in the studied area: Anaplasma phagocytophilum, Rickettsia helvetica, Babesia venatorum and B. divergens, Francisella tularensis, Borrelia miyamotoi, B. afzelii/valaisiana, B. garinii/lusitaniae and Bartonella spp. CONCLUSION: Our findings reinforce the conclusion that ticks are important vectors of pathogenic microorganisms in suburban forests and suggest that despite complex intra-annual relationships between tick densities and temperature, there is no evidence for a climate-associated increase in infection risk over the 7-year period. Rather, tick densities are likely to be strongly influenced by population density fluctuations in vertebrate host species and wildlife management. Further detailed studies on the impact of climate change on tick population densities are required.

Infection of Siberian chipmunks (Tamias sibiricus barberi) with Borrelia sp. reveals a low reservoir competence under experimental conditions.

Reservoir competence is a key parameter in understanding the role of host species in the epidemiology of multi-host-especially vector-borne-pathogens. With this aim in view, we studied the reservoir competence of the Siberian chipmunk (Tamias sibiricus barberi) recently introduced into Europe, for the multi-host tick-borne bacteria, Borrelia burgdorferi sl, the agent of Lyme borreliosis. T. sibiricus were experimentally exposed to bites from Ixodes ricinus ticks infected with Borrelia burgdorferi sensu stricto and Borrelia afzelii, with subsequent assessment of bacteremia and antibody responses. Borrelia was detected in chipmunk blood samples, ear biopsies and organ necropsies, and in nymphs used for xenodiagnosis (at one and six months after the initial chipmunk infection) via both serological and molecular methods. In total, eight out of twelve chipmunks showed evidence of infection by Borrelia sp., either by ELISA or PCR. Five chipmunks developed an immune response against the bacteria one month after infection. Borrelia infection in at least one organ was observed in seven animals at 14, 38, 93 or 178 days post-infection. Xenodiagnosis was positive for one chipmunk at 38 days, but no longer at 178 days post-infection. Four chipmunks remained uninfected, despite similar infection pressures to those observed in the field. Taken together, these results suggest that chipmunks can be infected through Borrelia-infected tick bites, and can transmit Borrelia to nymphs, but do not remain persistently infected.

IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection.

Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.

Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France.

BACKGROUND: In France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France. METHODS: We analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries. RESULTS: We found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands. CONCLUSIONS: The genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

Impact of feeding system and infection status of the blood meal on Ixodes ricinus feeding.

Artificial membrane feeding systems are effective tools for both tick rearing and studying tick-borne pathogen transmission. In order to compare the effects of the type of feeding system on tick engorgement, Ixodes ricinus ticks were either fed on an artificial membrane feeding system or on live mice. Sheep and chicken blood were used with the membrane system to assess the effects of blood origin on tick engorgement. To investigate the effects of blood meal infection on tick engorgement, ticks were either fed with Bartonella-infected or uninfected blood, both via membrane feeding and on mice. The proportion of engorged ticks, the duration of tick feeding, and the weight of engorged ticks were assessed. Feeding on the artificial system led to a longer duration of tick feeding and a lower proportion of engorged ticks than when fed on mice, however, the weight of engorged ticks was unaffected. The proportion and weight of engorged ticks, as well as the duration of feeding were not affected by blood origin. Feeding on an infected blood meal or on infected mice decreased the proportion and the weight of engorged ticks, but did not affect tick feeding duration.

Vector competence of the tick Ixodes ricinus for transmission of Bartonella birtlesii.

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

Questing ticks in suburban forest are infected by at least six tick-borne pathogens.

The role of Ixodes ricinus ticks in the transmission of pathogens of public health importance such as Borrelia burgdorferi s.l. is widely recognized and is suspected in several emerging vector-borne pathogens in Europe. Here, we assess prevalence rates of several endemic and emerging zoonotic pathogens in tick populations in an area of high human population density in France, to contribute to a risk assessment for potential transmission to humans. Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs) and low to moderate ones of Anaplasma phagocytophilum (~1%), spotted fever group Rickettsia spp. (~6%), Babesia sp. EU1 (~1%), Bartonella birtlesii (0.1%), and Francisella tularensis (!1%). Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases.

Prevalence of five pathogenic agents in questing Ixodes ricinus ticks from western France.

In Europe, Ixodes ricinus ticks are vectors of many emerging pathogens, including Borrelia burgdorferi sensu lato (sl), Anaplasma phagocytophilum, spotted fever group Rickettsia sp., Babesia sp., and very likely Bartonella sp. In this study, we looked for the presence of DNA of these microorganisms in 572 ticks from two forests in the west of France. DNA extraction and polymerase chain reaction (PCR) amplification were performed on individual nymphal, male, and female I. ricinus ticks. Amplification from 1 tick among the 572 samples (0.2%) resulted in PCR products with Bartonella-specific primers. Sequence analysis of the amplified fragment did not lead to species identification. Two ticks (0.3%) carried A. phagocytophilum-specific DNA. Eight ticks (1.4%) were positive with spotted fever group Rickettsia-specific primers, and all PCR fragments were related to Rickettsia helvetica. Thirty-five ticks (6.1%) were positive with B. burgdorferi sl-specific primers; the sequences were all related to Borrelia garinii or Borrelia afzelii, except one that was related to Borrelia carolinensis, a newly described species never reported in Europe so far. Thirty-five ticks (6.1%) carried Babesia sp. DNA. Female adults were more infected by B. burgdorferi sl than male adults. The prevalence of B. burgdorferi sl and Babesia sp. was significantly different between the two forests, with a higher prevalence of B. burgdorferi sl in ticks from the forest of Princé and a higher prevalence of Babesia sp. in ticks from the forest of Gâvre. To our knowledge, this is the first study that has detected all five pathogens in questing I. ricinus in the west of France and the first report of B. carolinensis DNA in ticks in Europe.

Swine infection with Trichinella spiralis: Comparative analysis of the mucosal intestinal and systemic immune responses.

The immune protective response developed by swine against Trichinella spiralis is not yet fully understood, particularly at the mucosal level. This study aimed to characterise intestinal immunity to T. spiralis by comparison with the systemic response in specifically pathogen-free pigs. For this purpose, the kinetics of cytokine and antibody production were assessed in the intestinal mucosa and serum of swine infected with T. spiralis for up to 60 days post-infection (dpi). An ex vivo model of jejunum mucosa culture was used to collect the supernatant as a source of antibodies (Abs). Mucosal antibodies were observed by Western blot from 15 dpi, while serum antibodies were expressed from 20 dpi. Both sources of antibodies initially recognized a 110 kDa protein, followed by the identification of 35, 43/46 and 55/59 kDa proteins. IgG1 and IgA antibodies were strongly expressed within the mucosa. The expression levels of Type 1 (IFN-gamma, IL-12), Type 2 (IL-4, IL-6), pro-inflammatory (TNF-alpha) and regulatory (IL-10, TGF-beta) cytokines were assessed by RT-PCR in the intestinal mucosa and spleen. Both IL-10 and IFN-gamma mRNA levels were increased in mucosa, whereas IL-6 and IL-12 mRNA were expressed in spleen. Taken together, these results demonstrated a mixed Type 1/Type 2 profile, the Type 2 profile being dominant in the mucosa.

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis.

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ISA 70 water in oil emulsion and Montanide IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.

Establishment and characterization of continuous hematopoietic progenitors-derived pig normal mast cell lines.

Mast cells (MCs) are tissue resident, hematopoietic stem cells-derived elements, distributed throughout the body. They are the pivotal mediating cells of allergic reactions. In addition, in mice, MCs play a critical role in the defense against several pathogens, such as bacteria, parasites and viruses. Whereas the biology of rodent and human MCs has been extensively studied using in vitro derived populations, the role of MCs in pigs has not yet been evaluated, given the very low availability of pure porcine MCs populations. In the present report, we describe an original method to obtain continuous factor-dependent normal pig MCs (PMC) lines from fetal hematopoietic progenitors. These Stem Cell Factor (SCF) and Interleukin-3- (IL-3)-dependent PMC lines retain their capacity to growth after conventional freezing methods and exhibit most of the morphological and biochemical properties of normal, although immature, MCs, including metachromatic granules containing sulfated polysaccharides, the expression of c-kit and high-affinity IgE receptors (FcepsilonRI), and the ability to store histamine that is released upon cross-linking of FcepsilonRI. In vitro derived PMC lines might thus be valuable tools to further investigate the reactivity of these elements towards several parasites frequently encountered in pig, such as, but not limited to, Ascaris suum, Trichinella spiralis or Trichuris suis, or towards antigens derived from these pathogens.