Replication and tissue tropisms of Crimean-Congo hemorrhagic fever virus in experimentally infected adult Hyalomma truncatum (Acari: Ixodidae).

Adult Hyalomma truncatum Koch ticks were inoculated intracoelomically with Crimean-Congo hemorrhagic fever (CCHF) virus to examine tissue tropisms of this virus in ticks. Virus was recovered from all 185 ticks examined between 2 and 42 d after inoculation with CCHF virus. Titers or virus recovered from unfed male and female ticks were essentially the same (means, 10(2.4) and 10(2.5) plaque-forming units [PFU] per tick, respectively). Infection rates and titers recovered per gland for both salivary glands and reproductive tissues (ovaries and testes) were positively associated with blood feeding; average titers were 10-fold greater in organs from fed compared with those from unfed individuals. In contrast, neither the tick's sex nor feeding status (unfed or engorged) affected the titer of virus recovered from hemolymph (mean, 10(1.6) PFU/microliters). Although virus was recovered from Malpighian tubules, midgut, muscle, and nervous tissues from nearly all of the ticks tested, viral titers were consistently low. The increase in viral titer associated with blood feeding by ticks appeared to be due primarily to proliferation of tissue (e.g., salivary gland and reproductive tissues), rather than to increased replication in tissue already present.     

Venezuelan equine encephalomyelitis virus infection in and transmission by the tick Amblyomma cajennense (Arachnida: Ixodidae)

To assess a possible role of ticks as the maintenance host for epizootic strains of Venezuelan equine encephalomyelitis (VEE) virus, laboratory experiments were conducted to determine if ticks could become infected, maintain, and transmit the virus. Larval and nymphal Amblyomma cajennense (F.) and larval Dermacentor nitens Neumann ticks were exposed to epizootic VEE virus (Trinidad donkey strain) by allowing them to feed on viremic guinea pigs (strain 13). In A. cajennense, transstadial transmission was observed from larvae to nymphs and adults. Horizontal viral transmission to a mammalian host was accomplished by nymphs. Infection rates in nymphs and adults were 2% (42/2,750) and 4% (9/244), respectively, afer ingestion of virus as larvae. Virus was detected in A. cajennense adult ticks for up to 171 d after infection in the larval stage. A cajennense, exposed as nymphs, ingested virus but did not become infected (0/164 after 10 d after taking an infective bloodmeal). No virus was detected in D. nitens 7 d after exposure. These findings suggest that A. cajennense potentially could be involved in an interepizootic maintenance cycle of epizootic VEE viral strains.     

Replication of Crimean-Congo hemorrhagic fever virus in four species of ixodid ticks (Acari) infected experimentally

The vector potentials of Hyalomma dromedarii Koch, H. impeltatum Schulze & Schlottke, H. truncatum Koch, and Rhipicephalus appendiculatus Neumann for Crimean-Congo hemorrhagic fever (CCHF) virus (IbAr 10200) were evaluated by intracoelomic inoculation. All three Hyalomma species became infected; infection rates ranged between 80 and 100% at 7-14 d after inoculation, and viral titers increased in unfed specimens almost 100 times above inoculation levels within the first week following infection. Only 40% of the R. appendiculatus became infected, and viral titers of unfed specimens increased less than 10 times above inoculation levels. The virus persisted to 153 d in unfed H. impeltatum. Viral titers were significantly higher in female than in male H. dromedarii, H. impeltatum, H. truncatum, and R. appendiculatus after blood feeding. Blood feeding had little effect on the viral titers of male Hyalomma species. However, the percentage of female and male ticks from which virus was recovered was significantly higher from fed ticks compared with unfed ticks. No virological evidence of transovarial transmission was found in greater than 78,000 first-generation progeny (larvae, nymphs, and adults) of inoculated female H. dromedarii, H. impeltatum, H. truncatum, and R. appendiculatus. All species transmitted CCHF virus to guinea pigs when allowed to feed at both 6 and 21 d after inoculation.     

African swine fever virus infection in the Iberian soft tick, Ornithodoros (Pavlovskyella) marocanus (Acari: Argasidae).

One thousand six hundred Ornithodoros (Pavlovskyella) marocanus Velu larvae were fed on a pig infected with African swine fever virus (titer: 10(7.4) HAd50/ml), and 1,600 larvae were fed on an uninfected pig. Ticks in each group were compared for mortality rates, mean time to death for ticks that died, mean time from feeding to either molting or eclosion, percentage of ticks that eclosed or molted, and the number of blood meals per nymph or instar. Cumulative virus-induced mortality for all immature stages (larvae to adult) of O. marocanus that had been fed as larvae on a pig infected with African swine fever was ca. 73% over a 390-d period. In contrast, less than 9% mortality was observed among ticks fed on uninfected pigs. Mean time to death for infected ticks was 15-87 d versus 10-17 d for uninfected ticks. Differences in the premolt period (number of days from blood meal to molt) between infected and control ticks were not observed. Mean premolt periods for larvae and first-, second-, third-, fourth-, and fifth-instar nymphs fed on pigs were 7, 9, 15, 11, 15, and 15 d, respectively. The majority of infected and all uninfected ticks required only one blood meal from pigs to molt. Mean weights for unfed second-, third-, and fourth-instar nymphs and males and females were 0.50, 0.67, 3.07, 3.63, and 5.91 mg, respectively.     

Study of the seasonal dynamics, life cycle, and host specificity of Amblyomma aureolatum (Acari: Ixodidae)

In the first part of this study, monthly infestation by ticks was evaluated on dogs from December 2000 to November 2002 in the rural area of Taia?upeba, S?o Paulo. Adults of Amblyomma aureolatum (Pallas) were found on dogs in all months, with a mean prevalence per month of 46.9 +/- 15.7% (range, 25-80%). The mean tick relative abundance per month was 2.4 +/- 2.7 ticks (range, 0.5-14 ticks), and the mean tick mean intensity per month was 4.7 +/- 4.2 ticks (range, 1.5-23.3 ticks). No A. aureolatum immature ticks were found on dogs. In the second part of this study, we studied the life cycle of A. aureolatum in the laboratory. We tested the suitability of six host species for the immature stages and dogs for the adult stage. Tick developmental periods were observed at different temperatures (23, 25, or 27 degrees C), always with RH >95%, which were satisfactory for all free-living developmental stages of the tick life cycle. Chickens and guinea pigs were the most suitable hosts for larvae and nymphs (recovery rates, 18.4-52.2%). Dogs were highly suitable for adult ticks (all females exposed to them were recovered and laid eggs) but were unsuitable for the immature ticks (recovery rates, 0-10%). Based on published host records for A. aureolatum, our results indicate that dogs and birds are primary hosts for adult and immature stages, respectively, of A. aureolatum in nature. In addition, wild guinea pigs are indicated as another potential primary host for immature ticks.     

Changes in the concentration of globulins in naive guinea pigs during feeding by the immature stages of Rhipicephalus evertsi evertsi (Acari: Ixodidae)

The weight of Rhipicephalus evertsi evertsi Neumann nymphs, which as larvae and nymphs completed the entire blood meal on the same individual guinea pig, was significantly lower than the weight of those that as larvae and nymphs fed on two separate naive hosts. Nymphs of the latter category spent 1 wk (from unfed larvae to unfed nymphs) on one animal before their transfer to the second host to complete the blood meal. The albumin concentration of the host blood decreased and was related to the time that the immature ticks spent on the hosts. The albumin/globulin ratio also decreased. The alpha 1 globulin fraction increased soon after the guinea pigs were infested with ticks. No further changes in the levels of the alpha 1 globulin fraction were observed with time. The concentration of serum beta globulins increased only in guinea pigs infested with immature ticks for the entire larval and nymphal feeding period. A second infestation of those guinea pigs with larvae of R. e. evertsi resulted in further increases in the levels of serum beta globulins. The relationships among serum beta globulin levels, the weight of engorged nymphs, and host immunity are discussed.     

The effect of virus-immune hosts on Thogoto virus infection of the tick, Rhipicephalus appendiculatus

Thogoto (THO) virus infections of Rhipicephalus appendiculatus ticks were examined using tick hosts immune to the virus. In the first set of experiments, ticks were infected by feeding on viraemic hamsters. Inter-stadial infection of THO virus was not affected when ticks ingested a virus-immune bloodmeal but there was an effect on persistence of the virus. The incidence of intra-stadial infection was reduced by at least 40% when nymphs partially fed on viraemic hamsters and completed their bloodmeal on a virus-immune guinea pig. When the reverse situation was examined--feeding on a virus-immune host and then a viraemic host--no difference was observed in the number of ticks infected. In the second set of experiments, uninfected ticks acquired virus by co-feeding with infected ticks on apparently non-viraemic guinea pigs. Non-viraemic transmission of the viruses was inhibited when the guinea pigs were immune to either the Sicilian (SiAr 126) or prototype (IIA) isolates of THO virus. The laboratory data indicate that virus-immune hosts may have a significant effect on the role played by ticks in the epidemiology of tick-borne viruses.     

African swine fever virus infection in the soft tick, Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae).

In total, 1,186 second instar Ornithodoros (Alectorobius) puertoricensis Fox second instars were fed on a pig when it had a viremia of 10(5.2) hemadsorption units (HAd50/ml) and 420 second-instar O. puertoricensis were fed on an uninfected pig. Subsequent blood meals for ticks in both groups were from uninfected pigs. The effects of African swine fever virus (ASFV) infection on O. puertoricensis populations were evaluated for the following parameters: mortality; mean time to death; percentage molted per instar; percentage molted to male, female, or subsequent instar; effects on duration of premolt period; and the number of blood meals per instar. The cumulative virus-induced mortality rate for all immature stages (second to fifth instar) of O. puertoricensis that had been fed as second instars on a pig infected with ASFV was 43.2%. In contrast, 23.1% mortality was observed among ticks fed on uninfected pigs. The mortality rate among third instars that fed on the viremic pig was 55.3% versus 4.8% among nymphs fed on normal pigs. One-third to more than one-half of all third, fourth, and fifth instars required at least two blood meals to molt. Mean premolt periods for second, third, fourth, fifth, and sixth instars fed on uninfected pigs were approximately 12, 15, 32, 22, and 14 d, respectively. Mean weights for unfed second to fifth instars, males, and females were: 0.6, 1.0, 1.5, 1.7, 1.5, and 3.1 mg per tick, respectively.     

Experimental transmission of eastern equine encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae)

We evaluated the potential for Ochlerotatus j. japonicus (Theobald), a newly recognized invasive mosquito species in the United States, to transmit eastern equine encephalitis (EEE) virus. Aedes albopictus (Skuse) and Culex pipiens (L.) were similarly tested for comparison. Ochlerotatus j. japonicus and Ae. albopictus became infected and transmitted EEE virus by bite after feeding on young chickens 1 d after they had been inoculated with EEE virus (viremias ranging from 10(7.0-8.7) plaque-forming units [PFU]/ml of blood). No Cx. pipiens (n = 20) had detectable levels of virus 14 d after feeding on an EEE-virus infected chicken with a viremia of 10(8.1) PFU per ml of blood. Depending on the viral titer in the donor chicken, infection rates ranged from 55-100% for Oc. j. japonicus and 93-100% for Ae. albopictus. In these two species, dissemination rates were identical to or nearly identical to infection rates. Depending on the viral titer in the blood meal, estimated transmission rates ranged from 15 to 25% for Oc. j. japonicus and 59-63% for Ae. albopictus. Studies of replication of EEE virus in Oc. j. japonicus showed that there was an "eclipse phase" in the first 4 d after an infectious blood meal, that viral titers peak by day 7 at around 10(5.7) per mosquito, and that virus escaped the mid-gut as soon as 3 d after the infectious blood meal. These data, combined with the opportunistic feeding behavior of Oc. j. japonicus in Asia and the reported expansion of its range in the eastern United States, indicate that it could function as a bridge vector for EEE virus between the enzootic Culiseta melanura (Coquillett)-avian cycle and susceptible mammalian hosts.     

Experimental transmission of Crimean-Congo hemorrhagic fever virus by Rhipicephalus evertsi evertsi (Acarina:Ixodidae)]

We have conducted experiments to assess the ability of Rhipicephalus evertsi evertsi tick to transmit the Crimean-Congo haemorrhagic fever (CCHF) and determine their reproductive cycle. The Rh. e. evertsi was infected by intracoelomic (transparietal and intra-anal) inoculation during the imaginal stases and by oral feeding on an infected viremic goat during imaginal and nymphal stases. The infection rate, transovarial and trans-stasial CCHF virus transmission were monitored for virus reisolation after suckling mice inoculation and the virus identified by ELISA and IFA for antigen detection. After intracoelomic inoculation, unfed adults had viral titers ranging from 0.67 to 2.9 log DL50/0.02 ml and had transmitted the virus to their vertebrate hosts by blood feeding. After 8 to 10 days of blood feeding duration, infection rates were respectively 36% and 100% for male and female ticks. In two instances out of seven transovarial transmission was observed and the virus reisolated from larvae of first generation. However, the virus was not detected after nymphal metamorphosis. After blood feeding on viremic goats, 71% of the nymphae were infected. After metamorphoses 22% of the males and 42% of the females had a low virus titer. Rh. e. evertsi appears to have a limited efficacy in transmitting and replicating the CCHF virus but must be not neglected as a potential vector during an epizootic manifestation.     

Experimental transmission of African swine fever virus by the tick Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae).

The soft tick Ornithodoros puertoricensis Fox has been found on the Caribbean island of Hispaniola (Haiti and the Dominican Republic) where African swine fever (ASF) was endemic from 1978 to 1984. To evaluate the vector potential of O. puertoricensis for African swine fever virus (ASFV), second-instar nymphs were experimentally infected by feeding on a viremic pig that was infected with the Dominican Republic isolate (DR-II) of ASFV. Subsequent infection rates and mean virus titers for individually triturated ticks were: second-instar nymph (95.4%, 10(4.38 +/- 0.85)), third-instar nymph (48.9%, 10(4.59 +/- 0.61)), male (46.7%, 10(4.36 +/- 0.61)), and female (35.3%, 10(4.38 +/- 1.09)). Infected ticks transmitted ASFV to susceptible pigs by bite 23, 85, 126, 160, 182, and 239 d after the infective blood meal. These findings show that ASFV is passed transstadially among O. puertoricensis and that O. puertoricensis can be an efficient vector of African swine fever virus.     

Experimental transmission of African swine fever virus by the soft tick Ornithodoros (Pavlovskyella) marocanus (Acari: Ixodoidea: Argasidae).

A total of 1,600 Ornithodoros (Pavlovskyella) marcocanus larvae were fed on a pig with a viremia of 10(7.4) HAd50/ml of African swine fever virus (ASFV). Infected larvae were sampled daily for 15 d, and nymphs were sampled at least once per instar until they became adults. Initial titers of 10(4.48) HAd50 per larva declined to 10(4.04) within 2 d. Larval titers reached a maximum of 10(6.0) HAd50 per larva 10 d after the infective blood meal. Nymphs of each instar were fed on a susceptible pig and in each case transmitted ASFV by bite. Virus titers for first to fourth instars ranged from 10(4.61) to 10(3.34) HAd50 per nymph. Transstadial survival occurred in subsequent first, second, third, and fourth instars with an 89% survival rate over 250 d. Approximately 30% of adult ticks that were infected as larvae remained infected and transmitted ASFV to susceptible pigs 588 d later. In addition, ASFV was recovered from the same adult ticks 655 d after the infective blood meal.     

Ticks parasitizing dogs in northwestern Georgia

From January 1998 through September 1999, 324 dogs in three northwestern Georgia counties were examined for ticks. Six species of ticks were recovered. The three most commonly collected ticks were the American dog tick, Dermacentor variabilis (Say) (310 male male, 352 female female; prevalence, 97%; mean intensity 2.1); the brown dog tick, Rhipicephalus sanguineus (Latreille) (118 male male, 119 female female, 38 nymphs; prevalence, 22%; mean intensity, 3.8); and the lone star tick, Amblyomma americanum (L.) (8 male male 26 female female, 2 nymphs; prevalence, 5%; mean intensity, 2.4). Other ticks recovered were Ixodes cookei Packard (3 female female); the Gulf Coast tick, Amblyomma maculatum Koch (2 female female); and the blacklegged tick, Ixodes scapularis Say (1 female). Another adult female specimen of I scapularis was recovered from a cat, further reinforcing that this medically important tick is present in northwestern Georgia.     

Laboratory studies on the life cycle of Amblyomma marmoreum (Acari: Ixodidae) on two different hosts.

A comparative study was made of the life cycle of the tortoise tick, Amblyomma marmoreum Koch, on tortoises and guinea pigs under laboratory conditions. At 25 degrees C and 85% RH with natural day length, duration of off-host stages (preoviposition, oviposition, incubation, and premolt) was similar for ticks fed on both hosts. Delay in preoviposition (up to 90 d) was observed in some gravid females. Larvae, nymphs, and adults had longer feeding periods on tortoises than on guinea pigs. Adult females fed on tortoises had greater engorgement wieght and ovipositional capacity than ticks fed on guinea pigs. It is suggested that the shortened feeding period of immature stages on mammalian hosts together with the occurrence of morphogenic diapause may be more important than previously recognized in determining whether the life cycle of A. marmoreum is completed in 1 or 2 yr under natural conditions.     

Pathogenesis of Lassa virus infection in guinea pigs.

A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.     

Temporal changes in outer surface proteins A and C of the lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice

The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.     

Influence of climate on the proportion of Ixodes ricinus nymphs and adults questing in a tick population

We studied the relationship between climate and Ixodes ricinus L. tick behavior by following every day the proportion of ticks questing in a tick population placed in polyamide mesh-delimited arenas in the field. Simultaneously, the phenology of the questing density of nymphs and adults was studied by sampling ticks in a close location. At any time during the year, the proportion of questing adults was significantly higher (mean 24%) than the proportion of questing nymphs (mean 12%). The proportion of questing nymphs and adults decreased stepwise with time. The proportion of questing adults partially recovered after each decrease. In contrast, the proportion of questing nymphs was strongly reduced during a single short period in June and did not recover even partially. Decrease in the proportion of questing ticks was strongly related either to a peak in saturation deficit or to a drop in maximal relative humidity. No increase in the proportion of questing nymphs was observed in the arenas during autumn, although an autumn peak of nymphs was observed at the sampling location close to the arenas. This suggests that the autumn peak of nymphs observed in nature was due to newly emerged spring-fed larvae and not to reactivated spring active nymphs.     

Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission

Rodents play a significant role in enzootic cycles of tick-borne pathogens, notably, in the northern hemisphere, tick-borne encephalitis virus and Lyme borreliosis spirochaetes. The relative numbers of nymphal and larval ticks feeding on rodents are crucial variables in determining the probability of rodent infection and the degree of amplification of infection prevalence in the tick population. Manipulation of the microclimate within quasinatural experimental arenas revealed that under increasingly dry conditions the numbers of unfed nymphal Ixodes ricinus L. questing in upper layers of the herbage decreased, whereas the rate of fat use and the numbers of nymphs feeding on small rodents, both increased. This is consistent with nymphs descending to the moist lower vegetation layers for water replenishment, where they would come into contact with small hosts. Very few larvae quested or fed on rodents under the dry conditions, but many more did so once the humidity increased, suggesting that larvae escape desiccation by becoming quiescent. The ratio of larvae to nymphs feeding on rodents thus increases with increasing humidity, contributing to the seasonal and geographical variation in disease transmission dynamics.     

The potential of Aedes triseriatus (Diptera: Culicidae) as an enzootic vector of West Nile virus

The susceptibility of Aedes triseriatus (Say) (Diptera: Culicidae) to low levels of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was determined and compared with that of Culex pipiens L. to assess the likelihood of its participation in an enzootic cycle involving mammals. Ae. triseriatus and Cx. pipiens were exposed to WNV by feeding on baby chickens with WNV serum titers ranging from 10(4.1 +/- 0.1) to 10(8.6 +/- 0.1) plaque-forming units (PFU)/ml and from 10(4.1 +/- 0.1) to 10(7.0) PFU/ml, respectively. Infection rates and 95% confidence intervals (CIs) of 8% (4, 14) and 25% (15, 38) occurred in Ae. triseriatus and Cx. pipiens after feeding on chickens with WNV titers of 10(4.1 +/- 0.1) PFU/ml and increased to 65% (49, 79) and 100% (72, 100) in Ae. triseriatus and Cx. pipiens after feeding on chickens with titers of 10(7.1 +/- 0.1) PFU/ml. The mean infection rate of Ae. triseriatus ranged from 97% (84, 100) to 100% (79, 100) after feeding on chickens with WNV titers of > or = 10(8.2) PFU/ml. The infectious dose (ID)50 values for Ae. triseriatus and Cx. pipiens were 10(6.5) (6.4, 6.7) and 10(4.9) (4.6, 5.1) PFU/ml, respectively. The combined estimated transmission rate of Ae. triseriatus at 14 and 18 d after feeding on chickens with a mean WNV titer of 10(8.6 +/- 0.1) PFU/ml was 55%. Although Ae. triseriatus is significantly less susceptible to WNV than Cx. pipiens, the susceptibility of Ae. triseriatus to WNV titers < 10(5.0) PFU/ml and its ability to transmit WNV suggest that Ae. triseriatus has the potential to be an enzootic vector among mammalian populations.