Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses

ABSTRACT. Experimental studies were undertaken to ascertain the vector potential of Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius for Thogoto (THO) and Dhori (DHO) viruses, candidate members of the Orthomyxoviridae. In the first set of experiments, ticks were infected orally by feeding on viraemic hamsters. THO virus replicated in R.appendiculatus and A.variegatum, persisted trans-stadially in both tick species, and was transmitted to susceptible hosts during feeding. In contrast, both R. appendiculatus and A. variegatum were refractory to per os infection by DHO virus. In the second set of experiments, engorged R. appendiculatus and A. variegatum nymphs were parenterally inoculated with DHO virus. The virus persisted transstadially in both tick species and was transmitted by bite to susceptible hosts. These results indicate that the midgut acts as a barrier to per os infection of R.appendiculatus and A. variegatum by DHO virus. However, when this barrier is bypassed, i.e. by parenteral inoculation, both R.appendiculatus and A.variegatum can serve as efficient vectors of DHO virus.     

Resistance and cross-resistance in guinea-pigs and rabbits to immature stages of ixodid ticks

Infestation of guinea-pigs and rabbits with larvae of any one of five species of ticks, Rhipicephalus appendiculatus Neumann, Rhipicephalus evertsi evertsi Neumann, Amblyomma hebrauem Koch, Amblyomma variegatum Fabricius and Ixodes ricinus L., conferred resistance in the animals when exposed to subsequent infestations with the same tick species. Resistance to infestations by other tick species was not observed.     

The impact of Ixodiphagus hookeri, a tick parasitoid, on Ambl yomma variegatum (Acari: Ixodidae) in a field trial in Kenya

In an experiment to investigate the effect of the tick parasitoid, Ixodiphagus hookeri, on tick numbers on cattle, 150 000 parasitoids were released over a period of 1 year in a field where ten cattle infested with multiple tick species were kept. Amblyomma variegatum was reduced from 44 to two ticks per animal while Rhipicephalus appendiculatus increased over the time of parasitoid release. During the time of release 51% of the nymphs of A. variegatum collected from the animals were parasitized. The recovery of the parasitoids after the releases were stopped was only 9%. The total numbers of A. variegatum remained low up to 1 year after the parasitoid release was stopped. This study gives an insight into how I. hookeri could be used strategically for the management of A. variegatum on small-scale farms.     

Ecological aspects of cattle tick control in central Zambia

In ecological studies in central Zambia, both climate and ecotype affected population dynamics of tick species. Below average rainfall for several years caused a suppression in numbers of Rhipicephalus appendiculatus Neumann adults. Reduction in rainfall leading to changes in grazing patterns is thought to have been responsible for an increase in numbers of Amblyomma variegatum Fabricius adults in a grassland habitat. There were reasonable correlations between numbers of each tick species on individual hosts over 1 year old. However, there were no relationships between numbers of ticks and bovine lymphocyte antigens (BoLA).     

Susceptibility of mosquito and tick cell lines to infection with various flaviviruses

The genus Flavivirus consists of more than 70 virus species and subtypes, the majority of which are transmitted by mosquitoes or ticks, although some have no known vector (NKV). The ability of these viruses to infect cultured cells derived from mosquito or tick species offers a useful insight into the suitability of such vectors to harbour and replicate particular viruses. We undertook a comparative study of the susceptibility of mammalian Vero cells, a clonal mosquito cell line (C6/36) and recently developed cell lines derived from the ticks (Acari: Ixodidae) Ixodes ricinus (L.) (IRE/CTVM18), I. scapularis (Say) (ISE6), Rhipicephalus appendiculatus (Neumann) (RAE/CTVM1) and Amblyomma variegatum (Fabricius) (AVL/CTVM17) to infection with 13 flaviviruses (and one alphavirus) using immunofluorescence microscopy and plaque assay techniques. The C6/36 mosquito cell line was infected by all the mosquito-borne flaviviruses tested but not by NKV viruses or tick-borne viruses, with the exception of Langat virus (LGTV). The tick cell lines were susceptible to infection by all of the tick-borne viruses tested, as well as two mosquito-borne viruses, West Nile virus (WNV) and the alphavirus, Venezuelan equine encephalitis virus (VEEV), but not other mosquito-borne viruses or NKV viruses.     

In vitro assays for repellents and deterrents for ticks: differing effects of products when tested with attractant or arrestment stimuli

Most in vivo and in vitro tests with repellents or deterrents against ticks have not considered which sensory channel is being targeted. We have recorded the responses of two hard tick species (Acari: Ixodidae) in vitro to determine if such products can disrupt the perception of an attractant in a repellent assay or the perception of an arrestment stimulus in a deterrent assay. Ethyl butylacetylaminopropionate (EBAAP), N,N-diethyl-methyl-benzamide (deet), permethrin and indalone were chosen to test their capacity to inhibit the attraction of Amblyomma variegatum Fabricius to its aggregation-attachment pheromone. Vapours of each test product plus those from a synthetic blend of the pheromone were delivered to the walking tick in an air stream on a locomotion compensator. Neither EBAAP, deet, permethrin nor indalone could inhibit attraction of A. variegatum even when each of the test products was delivered at 106 times the pheromone. Indalone did decrease the attraction of A. variegatum to the pheromone and induced repulsion of A. variegatum when presented on its own in the air stream. The effect of permethrin, a sodium channel blocker, was also tested in a deterrent assay measuring the arrestment of Ixodes ricinus (L.) adults on its own faeces and faecal constituents. Permethrin deterred arrestment at doses of 670 fg/cm2 to 67 ng/cm2, i.e. at levels five times lower than the dose of chemostimuli present in the arrestment stimulus. This sensitivity to permethrin suggests that it acts via the contact chemoreception channel.     

Tick infection rates with Borrelia Ixodes ricinus versus Haemaphysalis concinna and Dermacentor reticulatus in two locations in eastern Germany

Unfed nymphal Ixodes ricinus, Haemaphysalis concinna, and adult Dermacentor reticulatus were collected in two locations of Saxony in July and September 1991 by flagging. In July, the abundance of nymphal I. ricinus was about 2-3 times higher than that of nymphal H. concinna, a time of the year when nymphs of both species are reported to have a seasonal peak of activity. No D. reticulatus were flagged concurrently. In September, host-seeking activity of nymphal I. ricinus was again quite high as was that of adult D. reticulatus but only low numbers of nymphal H. concinna were collected. The flagged ticks were individually examined for Borrelia by an indirect immunofluorescence assay (I. ricinus: n = 414; H. concinna: n = 96; D. reticulatus: n = 116). The prevalence of Borrelia (probably B. burgdorferi) in I. ricinus varied from 12.1% to 21.0%. No borreliae were found in H. concinna. Of the examined D. reticulatus from one site (n = 97) 11.3% contained either B. burgdorferi or a related Borrelia. This may be the first finding of Borrelia in an Eurasian Dermacentor species.     

Repellency of MyggA® Natural spray (para-menthane-3,8-diol) and RB86 (neem oil) against the tick Ixodes ricinus (Acari: Ixodidae) in the field in east-central Sweden

In the field in south-central Sweden, we tested by randomised, standardised methodology the potential anti-tick repellent activity of two concentrations of MyggA Natural spray (containing PMD) (4.2 and 3.2 g/m2) and one of RB86 (with 70% neem oil containing azadirachtin) (3 g/m2) to host seeking nymphs of Ixodes ricinus. Each substance was applied separately to 1 m2 cotton flannel cloths. Nymphal ticks on the cloths, pulled over the vegetation, were recorded at 10-m stops. Nymphal numbers recorded differed significantly between treated cloths [4.2 or 3.2 g MyggA Natural spray/m2 and 3 g RB86/m2] and the untreated control (df = 3, chi2 = 112.74, P < 0.0001). Nymphal numbers also differed significantly among collectors (df = 3, chi2 = 15.80, P < 0.001). Repellency of treated cloths, i.e., 4.2 or 3.2 g MyggA Natural spray/m2 and 3 g RB 86/m2 declined from day 0 (i.e. the day of impregnation) to day 3 after impregnation from 77 to 24%, 58 to 16% and 47 to 0.5%, respectively. This study suggests that all three treatments have significant repellent activities against I. ricinus nymphs.     

Borreliae in Ixodes ricinus ticks feeding on humans

A total of 298 Ixodes ricinus (L.) ticks (Acari: Ixodidae) feeding on humans in the Czech Republic were tested for borreliae (Borrelia burgdorferi sensu lato) by darkfield microscopy between 1997 and 2003. A majority (68%) of the supplied I. ricinus ticks were nymphs, 25% were females and 7% were larvae. Overall, 20% of 74 examined females and 9% of 203 examined nymphs (but none of 21 examined larvae) were infected with borreliae. The proportion of ticks with a high infection load (>100 spirochetes) was 4% in females and 2% in nymphal I. ricinus. During the year, the highest numbers and proportions of infected nymphal and female ticks were taken from humans in June. Detection of borreliae in the ticks feeding on humans might be helpful in the prophylaxis of Lyme borreliosis.     

Density of deer in relation to the prevalence of Borrelia burgdorferi s.1. in Ixodes ricinus nymphs in Rambouillet forest, France

The Rambouillet Forest, a Lyme disease-endemic area near Paris, France, was surveyed from September 1994 to October 1995 to determine the risk periods and zones for humans. Firstly, during the period of Ixodes ricinus activity, abundance of nymphs is greater in spring than in autumn. Secondly, we observed significant variation in nymphal abundance between zones according to the density of cervids. The polymerase chain reaction (PCR) was used to detect DNA of Borrelia burgdorferi sensu lato in 461 unfed nymphs. DNA was detected in 38 nymphs (8.2%). By genospecific PCR based on the OspA gene, we detected the three pathogenic spirochetes with occurrences of 10.3, 31.1 and 58.6 for B. burgdorferi s.s., Borrelia garinii and Borrelia afzelii, respectively, indicating that B. afzelii is probably the main Borrelia species in the Rambouillet Forest. Finally, 11.5% of positive nymphs exhibited a double infection. Infection rates of I. ricinus nymphs by B. burgdorferi s.l. were not significantly different throughout the year for a given area, indicating that the risk periods of acquiring Lyme disease are mainly linked to nymph activity and correspond to spring and autumn. Likewise infection rates of nymphs were not significantly different between zones with a high density of deer (more than 100 animals per 100 ha) and zones with lower deer density (less than 20 animals per 100 ha). In addition to the role of deer as an amplifier of tick populations, these data indicate that zones with a high density of cervids should be considered as higher risk areas. © Rapid Science Ltd. 1998     

Four serine proteinase inhibitors (serpin) from the brown ear tick,Rhiphicephalus appendiculatus; cDNA cloning and preliminary characterization

While development of an anti-Boophilus microplus vaccine is advanced and practical, work on other economically important ticks such as Rhipicephalus appendiculatus is still in its infancy. Guess PCR primers, designed from a consensus amino acid sequence (NAVYKFG) motif were used with rapid amplification of cDNA ends (RACE) to clone four cDNAs encoding serine proteinase inhibitors (serpin) from the brown ear tick Rhipicephalus appendiculatus. The four genes designated as R. appendiculatus serpin (RAS) -1 to -4 encode polypeptides of 378, 380, 398 and 486 amino acids long, respectively. Sequence comparison of RAS-1 to -4 predicted amino acid sequences to the serpin-like hypothetical protein from Ixodes ricinus (Leboulle et al., 2002) revealed closer structural similarities among tick serpins. Expression analysis by RT-PCR showed that RAS-1 to -4 are expressed in other tick organs in addition to salivary glands and midguts. Except for RAS-3 whose expression level appears to be equivalent in all tick organs, RAS-1, -2 and -4 are predominantly expressed in the salivary glands. We have discussed our findings with reference to development of vaccines against R. appendiculatus.     

Molecular individuality and adaptation of the tick Rhipicephalus appendiculatus in changed feeding environments

The tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae) naturally infests many host species. However, the mechanisms that enable it to feed on such a wide range of hosts are unclear. One possibility is that a tick population maintains molecular (genotypic and/or phenotypic) diversity among individuals such that individuals vary in their competency in taking bloodmeals under different feeding conditions. As a first step in testing this hypothesis, we showed that the polymorphism of salivary gland proteins, previously demonstrated in unfed ticks, was maintained during feeding on guinea-pigs. We then compared feeding performance under standard laboratory rearing conditions: one instar (adults or nymphs) feeding on guinea-pigs, with three changed conditions: (1) two instars (adults and nymphs) feeding together on guinea-pigs; (2) one instar (adults or nymphs) feeding on hamsters; and (3) two instars (adults and nymphs) feeding together on hamsters. The mean engorged weight of adult females was significantly reduced under all changed conditions, indicating that most of the adult individuals were significantly challenged by the changed conditions. However, some individuals achieved successful engorgement, indicating competence to the changed condition, and demonstrating variation in adaptive ability among individuals. Engorged females produced egg masses positively correlated to the engorged weights. More interestingly, the correlation coefficient (R) increased when feeding condition was changed. This may lead to more efficient selection for population adaptation under the changed conditions. As the feeding success of ixodid ticks depends on the efficiency of the cocktail of immunomodulatory saliva, the relevance of the polymorphism of salivary gland proteins and host adaptation is discussed.     

CCR5 regulates high dose oral tolerance by modulating CC chemokine ligand 2 levels in the GALT

Oral tolerance is an immunomodulatory mechanism used by gut tissues to induce systemic tolerance to ingested proteins. In models of disease, such as experimental autoimmune encephalomyelitis, oral tolerance has been used to protect against paralysis induced by immunization with myelin proteins. Previous work in our laboratory has shown a role for the chemokine, CCL2, and its receptor in the induction of high dose oral tolerance. In the present study, we report that two CCR5 ligands, CCL4 and CCL5, are expressed in gut tissues after Ag feeding. CCR5(-/-) mice were unable to be tolerized by feeding a high dose of Ag and were not protected from developing experimental autoimmune encephalomyelitis. Moreover, CCR5(-/-) mice did not display cytokine deviation as normally seen after high dose oral Ag. Using a selective CCR5 antagonist, methionine-RANTES, CCL2 expression was inhibited, resulting in enhanced IL-12 production and the inability for mice treated with methionine-RANTES to become orally tolerized. This current study suggests that CCR5 ligands may function to modulate CCL2 levels in the gut after Ag feeding, promoting a cellular environment that favors tolerance rather than immunity.     

Autochthonous and migratory birds as a dispersion source for Ixodes ricinus in southern Italy

The present study was carried out in a protected wooded area, which is part of the Parco Regionale Gallipoli Cognato Piccole Dolomiti Lucane, one of the most important ecological reserves in southern Italy. From April 2010 to April 2011, 212 birds, comprising 22 species from 12 families, were captured and examined for ticks. A total of 75 (35.4?%) birds were found infested by ticks, with 451 ticks being collected. All ticks were identified as Ixodes ricinus, of which 241 (53.4?%) were larvae and 210 nymphs (46.6?%). The highest intensity of infestation was found in April 2010, when 117 ticks were retrieved on 25 birds. No ticks were found on birds (n?=?5) netted in December 2010. High infestation rates were recorded on blackbirds (Turdus merula) (90 %; 29 birds examined) and on mistle thrushes (Turdus viscivorus) (100?%; 2 birds examined). The highest intensity of infestation by larvae was found on wrens (5.6 larvae/bird) and by nymphs on mistle thrushes (11.5 nymphs/bird). Temperature and number of hours of light showed to influence the activity of larvae and nymphs. These data support the notion that birds may be responsible for the heterogeneous distribution of I. ricinus in Europe, thus playing a role in the epidemiology of certain tick-borne pathogens.     

Salivary gland extract from Ixodes ricinus tick polarizes the cytokine profile toward Th2 and suppresses proliferation of T lymphocytes in human PBMC culture.

Tick salivary gland extract (SGE) was previously shown to inhibit murine T cell proliferation. In mice, SGE has an inhibitory effect on Th1 and a stimulatory effect on Th2 cytokine elaboration. In the present study, tick-mediated immunomodulation of human T cell proliferation and cytokine elaboration was analyzed using human peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A (Con A) or lipopolysaccharide (LPS). Using flow cytometry, tick saliva-induced changes were investigated in human mononuclear cell subpopulations. SGE from Ixodes ricinus dose-dependently inhibited human T cell proliferation. This finding supports the flow cytometry data, showing that the percentage of Con A-activated HLA-DR-CD3+ T lymphocytes and CD4+ CD8+ double-positive T cells decreased after SGE treatment. SGE significantly inhibited the in vitro production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) secreted by Th1 lymphocytes. In contrast, the elaboration of IL-4, IL-6, and IL-10 secreted by Th2 lymphocytes was significantly stimulated by I. ricinus SGE. Similarly, the production of both IL-1alpha and IL-1beta was significantly stimulated after SGE treatment. These data indicate that the tick-induced immunomodulatory events in humans are similar to those previously described in a murine model.     

Questing activity of Rhipicephalus appendiculatus (Acari: Ixodidae) nymphs: a random process?

Abstract.  In Zambia, an experiment under quasi-natural conditions indicated that Rhipicephalus appendiculatus nymphs react to prevailing climatic conditions. Higher temperatures and higher vapour pressure deficits lead to decreased activity. The majority of nymphs (>75%) were recorded at ground level. Simulations showed that larval phenology and temperature during the nymphal premoulting period largely explain the seasonal abundance patterns of nymphs, as observed on cattle, given the absence of a behavioural diapause. Consequently, the effect of climate, as observed in our studies, is masked. However, the results of the present study indicate that daily climatic conditions probably have a much larger effect on the transmission dynamics of Theileria parva . The vertical distribution of questing instars is a function of temperature and humidity. In years of unfavourable conditions, nymphs might feed mainly on hosts other than cattle, and this could govern the infection prevalence in the adult population. This suggestion is supported by previous epidemiological studies.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

Effect of tick control on liveweight gain of cattle in central Zambia

Field trials were carried out during 1982-86 in two different ecotypes in central Zambia to determine the impact of tick control on the liveweight gain (LWG) of cattle. During the first 2 years of the trial a diamidide acaricide (Amitraz) sprayed at weekly intervals caused periodic depression in LWG in young animals. Thereafter a pyrethroid acaricide (cypermethrin) was used. During the 1984-85 tick season infestations of 50-120 adult Amblyomma variegatum Fabricius in untreated animals caused significant reductions in LWG. From the differences in LWG between treated and untreated cattle, the induced loss in LWG was estimated at 46-61 g per engorged female A.variegatum.     

Population dynamics of ticks on indigenous cattle in a pastoral dry to semi-arid rangeland zone of Uganda

Studies on seasonality and population dynamics of ticks on indigenous cattle and their crosses (calves) were carried out in Buruli Ranching Scheme, Nabiswera, Luwero district of Uganda on three treatment groups of animals: group 1 (twice a week dipping), group 2 (once a month dipping) and group 3 (no tick control). During this study, four major species of ticks of economic importance were recorded in decreasing order of abundance: Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Amblyomma variegatum and Boophilus decoloratus. Of these ticks, a seasonal pattern of activity was only observed in R. appendiculatus ticks, with peak activities occurring during rainy seasons. Greater numbers of ticks were recorded on cows than calves in the three treatment groups, with the exception of A. variegatum where the reverse occurred. The mean numbers of ticks per animal were highly significantly different (p < 0.01) when group 1 animals were compared with group 2 and 3 animals. However, no significant differences (p > 0.05) were observed in mean tick numbers between group 2 and 3 animals. Highly significant differences (p < 0.01) were observed in mean tick numbers on cows and calves (more than 12 months old) in different calving seasons. The state of lactation only affected tick counts on cows in group 1; significantly more ticks (p < 0.01) were observed in lactating than non-lactating cows. Furthermore, significantly greater (p < 0.05) numbers of ticks (with the exception of B. decoloratus) were recorded during the second year of study (March 1992-May 1993) than the first year (January 1991-February 1992), despite lower rainfall during the former period.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.