Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log₁₀ plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.     

Transmission of louping ill virus between infected and uninfected ticks co-feeding on mountain hares

Abstract. Most of the data on oral infection of ticks by louping ill virus have been obtained from experiments in which animals were infected by syringe inoculation with infectious material. Using infected ticks to mimic the natural situation, we have demonstrated that louping ill (LI) virus transmission can occur from infected to uninfected Ixodes acinus feeding in close proximity on mountain hares (Lepus timidus). Under these conditions the hares developed either low or undetectable viraemias. Highest prevalence of LI virus infection was observed in recipient nymphs which had fed to repletion between days 3 and 7 post-attachment of virus-infected adults; following engorgement, 56% of nymphs acquired virus. These results demonstrate the efficient transmission of LI virus between co-feeding ticks on naive mountain hares. However, when ticks were allowed to co-feed on virus-immune hares a significant reduction in the frequency of infection was observed. Neither red deer (Cervus elaphus) nor New Zealand White rabbits supported transmission of LI virus. The significance of virus transmission between cofeeding ticks on LI virus epidemiology is discussed.     

Histological examination ofBorrelia burgdorferi infections in unfedIxodes ricinus nymphs

Borrelia burgdorferi, the agent of Lyme borreliosis, is vectored in Europe byIxodes ricinus. In unfed ticks, the spirochaete resides primarily in the midgut, but a low percentage (5.5%; 4/73) of naturally infected ticks may present a systemic infection involving organs such as the salivary glands and central ganglion (Burgdorferet al., 1983). In this study, we examined 79 unfed nymphs collected in two sites in Switzerland (Neuchatel and Aarberg), and 35 unfed nymphs which were fed as uninfected larvae on 3 infectedApodemus sylvaticus mice. Dieterle silver staining was used to visualize the spirochaetes in the ticks. Of the unfed field-collected nymphs, 21/79 (27%) were infected of which 2/21 (10%) had systemic infections. Taking account of the site of collection, we observed that 0/12 ticks from Neuchatel were systemically infected whereas 2/9 (22%) from Aarberg had a disseminated infection. Out of the 35 unfed nymphs examined after an infectious blood meal on rodents, 14 (40%) were infected and 2 (14%) had a disseminated infection. A total of 4/35 (11%) unfed infected nymphs presented a systemic infection which represents a higher percentage than previously described. The presence of spirochaetes in salivary glands of systemically infected ticks before the initiation of feeding may reduce the time delay generally recorded for the tick-borne transmission ofB. burgdorferi.     

Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods

Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non-viraemic guinea-pig. This phenomenon has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea-pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea-pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

Plant smells---important determinants in the behavior and rate of development of ixodid disease vectors]

Experiments with nymphs of Ixodes persulcatus and Dermacentor marginatus have shown that the rate and degree of engorgement, dropping off from the mouse, metamorphosis longevity and weight of emerging imagoes change under plant odour influence. The influence of acetone extract vapours of pepper, poplar buds, linden and aspen was tested. Under the influence of the vapour, acting as an attractant for the adults, engorgement weight of their nymphs and the weight of imagoes emerging from them increased; attractants change the nymph-imago metamorphosis duration as well. Repellent acting odours, as a rule, produce an opposite reaction. The phenomenon of the inversion of the infected D. marginatus reaction to the odour is proved in nymphs. Attractant for naive adult ticks (poplar buds) increased the time of feeding and weight of nymphs in comparison with the control. Just the opposite: nymphs infected with tick-borne encephalitis (TBE) virus produce an opposite reaction--weight decrease under influence of poplar buds as the repellent. Importance of the above phenomena for the ticks distribution and TBE epizootiology is discussed.     

Feeding patterns of immature stages of Hyalomma truncatum and Hyalomma marginatum rufipes on different hosts

In this study we examine the feeding patterns of immature stages of Hyalomma truncatum and Hyalomma marginatum rufipes ticks on different hosts. Larvae of H. truncatum developed through a three-host pattern on two species of field mice, Rhabdomys pumilio and Lemniscomys rosalia. On guinea-pigs, both Hyalomma species followed a mixed two-host and three-host pattern, with the latter route being preferred, since more than 70% of the fully fed larvae dropped off from their hosts. H. truncatum was a two-host tick on rabbits. Larvae of H. marginatum rufipes did not prefer R. pumilio and L. rosalia as hosts. On guinea-pigs, H. marginatum rufipes immatures showed a mixed two-host and three-host pattern with a bias towards the three-host life cycle, since approximately 58% of the fully fed larvae dropped off. On rabbits, H. marginatum rufipes was exclusively a two-host tick. Mean engorgement weights and blood quantities ingested by H. truncatum nymphs that developed through a three-host pattern on mice were significantly higher (p<0.0001) than for those that developed through a two-host pattern on guinea-pigs and rabbits. For H.marginatum rufipes, there were no significant differences (p>0.05) between engorgement weights of nymphs that developed through two-host and three-host patterns. However, there were significant differences (p<0.0001) in blood quantities ingested by nymphs of this tick species following feeding on different hosts.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Kinetics of ingested host immunoglobulin G in hemolymph and whole body homogenates during nymphal development of Dermacentor variabilis and Ixodes scapularis ticks (Acari: Ixodidae)

Patterns in the utilization of host immunoglobulin G (IgG) during nymphal development differed between Dermacentor varibilis (Say) and Ixodes scapularis Say ticks. In unfed nymphs of D. variabilis, host IgG was readily detectable in both hemolymph and whole body homogenates. In unfed nymphs of I. scapularis, host IgG was absent in hemolymph and at very low concentrations in whole body homogenates. Host IgG in unfed nymphs was undoubtedly the remnants of IgG acquired during the larval bloodmeal that persisted through metamorphosis to the nymphal stage. In both tick species, host IgG crossed the midgut into the hemocoel during the latter phases of engorgement. Concentrations of host IgG in I. scapularis declined considerably after replete nymphs molted to the adult stage. In contrast, concentrations of host IgG in D. variabilis remained elevated throughout metamorphosis to the adult stage. When larval D. variabilis were fed on a rat, then 2 months later as nymphs on a rabbit, the rat IgG (“old IgG”) present in unfed nymphs was totally replaced by rabbit IgG (“new IgG”) within 2 d of nymphs attaching to the rabbit. Presumably, the old IgG acquired from a previous bloodmeal was secreted via saliva into the new host. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age-related cannibalism and horizontal transmission of a nuclear polyhedrosis virus in larval Spodoptera frugiperda

1. Experiments were carried out to investigate the incidence of cannibalism throughout the larval development of the noctuid moth Spodoptera frugiperda, and to examine the risk of infection from consuming conspecifics infected with a nuclear polyhedrosis virus (SfNPV). 2. Cannibalism was observed commonly even when food was not limiting, but occurred more frequently at low food quantities and/or high rearing densities. The sex of the larvae had no effect on the incidence of cannibalistic behaviour, however the probability of cannibalism occurring was affected by larval stage. The frequency of cannibalism was significantly higher among fifth- and sixth-instar larvae than among earlier instars, and larvae were more likely to consume younger conspecifics than larvae of the same stage. 3. Fifth-instar larvae offered fourth-instar victims fed equally on healthy larvae, virus-infected larvae (2 days post-infection), uninfected corpses, and virus-killed corpses (6 days post-infection). Horizontal transmission of SfNPV was only recorded in larvae offered virus-killed corpses, however, and total mortality in this treatment was only 32%. 4. In a similar experiment, fourth-instar larvae avoided cannibalising virus-killed corpses. Horizontal transmission of SfNPV was recorded in fourth-instar larvae that consumed 2-day post-infected larvae. The low incidence of cannibalism observed in fourth-instar larvae, however, suggests that this is unlikely to provide an important route for the transmission of SfNPV.     

Ability of transovarially and subsequent transstadially infected Ixodes hexagonus ticks to maintain and transmit Borrelia burgdorferi in the laboratory

In a previous study, transstadial and transovarial survival of Borrelia burgdorferi in Ixodes hexagonus and transmission to laboratory mice via the bite of infected females were demonstrated. Here, we report the ability of I. hexagonus progeny infected transovarially to maintain and transmit the spriochaete to the host.Ticks were examined for spirochaetes by direct immunofluorescence antibody test. I. hexagonus larvae derived from the parental transstadially infected females were fed on two white mice: 21/54 (38.9%) of these ticks examined as unfed nymphs were infected. I. hexagonus nymphs were fed on three white mice and examined for spirochaetes after moulting as adults: 7/25 (28%) were found to harbour the spirochaete. The success of B. burgdorferi transmission to the mice by larval and nymphal I. hexagonus was determined by xenodiagnosis using I. ricinus larvae: 20/50 (40%) and 30/99 (30.3%) of the I. ricinus larvae fed on the mice infected by I. hexagonus larvae and nymphs respectively became infected.This study shows that B. burgdorferi can be maintained through transovarial and subsequent transstadial transmissions in I. hexagonus.     

Molecular evidence for trans‐stadial transmission of Anaplasma platys by Rhipicephalus sanguineus sensu lato under field conditions

This study investigated the transmission of Anaplasma platys by Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae). Engorged nymphs (n = 404) removed from A. platys‐infected dogs were incubated at 28 °C until moulting. Unfed adults were obtained and divided into 48 pools. Unfed ticks collected from the grounds of the dog shelter, comprising 1800 larvae (n = 18 pools), 3100 nymphs (n = 62 pools) and 85 adults (n = 10 pools, including three male and seven female pools) were sorted into 90 pools. All pools were screened by polymerase chain reaction for the 16S rRNA gene of A. platys. Of 48 pools of unfed adults obtained from engorged nymphs, 12 were positive for A. platys; the infection rate maximum likelihood estimate (MLE) was 3.36 [95% confidence interval (CI) 1.84–5.68]. Anaplasma platys was detected in five of 23 male pools (MLE 2.82, 95% CI 1.06–6.20) and seven of 25 female pools (MLE 3.83, 95% CI 1.72–7.57). Of seven pools of unfed adult females collected from the shelter grounds, one was positive for A. platys (MLE 1.74, 95% CI 0.11–8.22). Among 62 unfed nymph pools, eight were infected with A. platys (MLE 0.27, 95% CI 0.13–0.52). No A. platys DNA was detected in the larva pools. The present results reveal molecular evidence for the trans‐stadial transmission of A. platys by R. sanguineus s.l.     

Transmission of a nuclear polyhedrosis virus inHeliothis zea [Lep.: Noctuidae] larval populations on soybean

Transmission of a nuclear polyhedrosis virus (NPV) inHeliothis zea (Boddie) on soybean was examinated. Artificial infestations ofH. zea were established at densities of 6.5, 19.5 and 58.5 larvae/row-m. Additional larvae infected to die from NPV in the 2nd stage were released into subplots to simulate 5 and 25% mortality levels. Virus transmission from infected to noninfected larvae was correlated with the initial incidence of infection in the population but not the density of larvae/row-m. Deposition of virus on plants from cadavers of larvae that died of virus infection was correlated with the initial incidence of infection in the populations and the density of larvae/row-m. After pupation of larvae in the 1st population, noninfected larvae only were again released to examine transmission of viral inoculum remaining on plants and soil. The percent mortality of larvae collected from the 2nd release was low and did not differ significantly between treatments. The concentration of virus on foliage and in soil after the 2nd release was directly correlated with density of larvae/row-m but not the incidence of infection within the population in the 1st release. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement N^o 82 CRSR-2-1000.     

Mixed infection by tick-borne encephalitis virus and Borrelia in ticks

To investigate the relationships between tick-borne encephalitis (TBE) virus and the bacterial spirochaete Borrelia burgdorferi sensu lato in vectors with mixed infections, unfed adult Ixodes persulcatus ticks were collected by flagging from vegetation in southern-taiga forests of the Pre-Urals region of Russia where both infections circulate sympatrically. Prevalences of TBE and Borrelia infections in a total of 4234 ticks were compared over 5 years. No significant differences were revealed between the prevalence of Borrelia infection in ticks with and without TBE virus (29.4+/-7.8% vs 23+/-3.6%), or between the prevalence of TBE virus infection in ticks with and without Borrelia (24.0+/-6.6% vs 18.4+/-3.4%). In ticks with mixed infection (40/689 = 5.8%), concentrations of TBE virus and Borrelia were not significantly correlated with one another. Field observations showed parallel trends in the prevalence of these pathogens in tick populations from year to year (1993-1997) indicating that, in I. persulcatus with mixed infection, Borrelia and TBE virus do not seem to interfere with each other and are apparently not involved in any antagonistic relationships.     

Developmental and reproductive patterns of Triatoma brasiliensis infected with Trypanosoma cruzi under laboratory conditions

The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.     

Investigating the horizontal transmission of the Cydia pomonella granulovirus (CpGV) in a model system

Repeated applications of Cydia pomonella granulovirus (CpGV) can effectively control the codling moth (CM) in apple orchards. However, it is still unknown whether horizontal transmission of the virus from infected to uninfected larvae contributes to the efficacy of the virus insecticide. Horizontal transmission of CpGV was assayed using detached apples. In experiments using artificially applied virus dots on the apple’s surface or infected CM larvae as virus inoculum, it was found that the likelihood of infection of healthy CM larvae relied mainly on the larval behavior. The amount of virus inoculum, either applied artificially or produced by the infected larvae, impacted the infection rate only to a small degree. In the experiments, CM larvae exhibited a strong preference in entry sites, increasing the chance for horizontal transmission. Depending on the experimental design, horizontal transmission rates of about 40% were observed in laboratory assays.     

Density-related variation in vertical transmission of a virus in the African armyworm

Larvae of the African armyworm, Spodoptera exempta, are darker and more resistant to baculovirus infection when reared in groups (gregarious form) compared to being reared singly (solitary form). Lepidoptera that survive virus challenge as larvae could potentially retain a sublethal virus infection which is then transmitted vertically to the next generation. Here we examine whether gregarious and solitary forms of the armyworm differ in the costs of surviving virus infection and in their capacity to transmit an active baculovirus infection to their offspring. Pupae of larvae reared gregariously that survived virus challenge weighed significantly less than uninfected individuals, but this was not so for those reared solitarily. This did not, however, translate into differences in fecundity, at least under laboratory conditions. As found in previous studies, pre-oviposition period was shorter for solitary than gregarious insects, and it was also shorter for females that had been challenged with virus as larvae. Both the prevalence of egg batches containing larvae that died from nucleopolyhedrovirus (NPV) infection and the proportion of infected larvae within each egg batch were significantly increased (approximately doubled) when parental moths were previously challenged with the virus during their larval state. This demonstrates that horizontal transmission in one generation can elevate vertical transmission to the next generation. Moreover, prevalence of overt infection in the offspring generation was two to three times greater when parental moths were reared solitarily as larvae than when reared gregariously. Disease prevalence and proportional infection were both independent of the sex of the infected parent and whether or not the egg batch was surface-sterilized to remove potential contaminants. This suggests that the eggs are infected internally (transovarial) rather than externally (transovum). These results help to shed light on the observed temporal pattern of virus epizootics in eastern Africa.     

Role of small mammals in the ecology of Borrelia burgdorferi in a peri-urban park in north coastal California

The role of small mammals other than woodrats in the enzootiology of the Lyme disease spirochete, Borrelia burgorferi, was assessed in a peri-urban park. Mammals were collected monthly from September through to April. Following tick removal, the animals were tested for B. burgdorferi by culture of ear-punch biopsies. Larvae and nymphs that were intermediate in morphology between Ixodes spinipalpis and Ixodes neotomae occurred on several species of rodents (Peromyscus truei, Peromyscus californicus, Microtus californicus, Rattus rattus and Reithrodontomys megalotis) and the brush rabbit (Sylvilagus bachmani). Morphometric analyses of these I. spinipalpis-like ticks and the offspring from two I. neotomae females from this site suggest that I. neotomae may be conspecific with I. spinipalpis. Borrelia burgdorferi was isolated from eight out of 109 (7.3%), three out of 16 (18.8%), two out of 38 (5.3%) and two out of six (33.3%) P. truei, P. maniculatus, M. californicus and R. rattus, respectively. One brush rabbit yielded the first isolate of B. burgdorferi from a lagomorph in western North America. This isolate and three others derived from unfed I. spinipalpis-like nymphs failed to produce infection when inoculated intradermally into 11–12 P. maniculatus each. Likewise, no spirochetes were detected in 420 Ixodes pacificus nymphs derived from larvae fed on animals inoculated with these isolates. An additional isolate, derived from an I. spinipalpis-like nymph, was recovered by ear-punch biopsies from five out of 12 (42%) needle-inoculated P. maniculatus. However, spirochetes were not detected in 20 I. pacificus nymphs fed as larvae on each of five mice (two infected and three uninfected) inoculated with this isolate. We conclude that brush rabbits and several species of rodents besides woodrats may contribute to the maintenance of B. burgdorferi because they harbour the spirochete and are fed upon by competent enzootic and bridge vectors.     

Rates of bluetongue virus transmission between Culicoides sonorensis and sheep

Abstract.  Two experiments were undertaken to estimate the transmission rates of bluetongue virus (BTV) serotype 1 between a biting midge vector, Culicoides sonorensis (Wirth & Jones) (Ceratopogonidae), and a natural host, sheep. In an experiment to measure the transmission rate from vector to host (V→H), six batches of one, five and 20 intrathoracically infected midges were fed on a total of 18 bluetongue (BT)-naïve sheep. The sheep were then monitored for 21 days for clinical signs of BT, viraemia and antibody response. All sheep fed on by five or 20 midges and five of six sheep fed on by just one midge showed signs of BT, were viraemic and developed antibody. The sixth sheep fed on by a single infected midge did not show signs of BT or have detectable viraemia; it did, however, develop a weak antibody response. A bite from a single infected midge is therefore able to transmit BTV to naïve sheep with 80–100% efficiency. Sheep fed upon by larger numbers of infected midges took less time to reach maximum viraemia and developed stronger antibody responses. Sheep exposed to greater amounts of BTV in feeding midges developed a higher level of viraemia and stronger antibody responses. In a second experiment to measure the transmission rate from host to vector (H→V), batches of up to 500 uninfected female C. sonorensis fed every 1–2 days on two experimentally infected sheep during the course of infection. Of 3929 engorged midges that were individually titrated after surviving the extrinsic incubation period, only 23 (0.6%) were infected with BTV. Viraemia in the sheep extended for up to 19 days post-inoculation. No infected midges, however, were detected from 14 days post-infection.