Comparative infections of epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus in Amblyomma cajennense (Acari: Ixodidae).

To compare the potential for an enzootic or an epizootic strain of Venezuelan equine encephalomyelitis (VEE) virus to infect Amblyomma cajennense (F.), larval ticks were fed on guinea pigs (strain 13) inoculated with an enzootic viral strain of variant I-E (68U201) or on guinea pigs inoculated with an epizootic strain of variant I-A (Trinidad donkey). Peak viremias were 10(5.2) plaque-forming units (PFU)/ml and 10(7.3) PFU/ml in guinea pigs infected with enzootic and epizootic viral strains, respectively. Ticks feeding on enzootic- and epizootic-infected hosts had viral titers of 10(2.5) and 10(3.9) PFU per tick, respectively, at drop-off. Although epizootic virus was recovered from 98% (127 of 130) of larval ticks up to 16 d after drop-off, enzootic virus was recovered from 95% (19 of 20) at drop-off (mean titer, 10(2.5) PFU per tick), with recovery rates declining rapidly to 2 of 10 (mean titer, 10(1.4) PFU per tick) by 16 d after drop-off. Transstadially transmitted epizootic virus was found in 0.4% (12 of 2,950) of unfed nymphs (mean titer, 10(2.8) PFU per tick) 63 d after drop-off, 1% (5 of 521) fed nymphs 69 d after drop-off, and 1% (4 of 400) of unfed adults (mean titer, 10(3.6) PFU per tick) 106 d after drop-off. No enzootic virus was recovered from 4,600 unfed nymphs tested 63 d after drop-off.     

Experimental infection of Hyalomma marginatum ticks with West Nile virus

To define the possible role of Hyalomma marginatum ticks in the transmission of West Nile virus (WNV) in Portugal an experimental infection was established. Ticks were fed on viremic rabbits previously infected with WNV. In different developmental stage of H. marginatum virus isolation and detection of viral antigen and viral RNA were attempted. The oral infection rates were 3%, 33% and 75% for engorged larvae, nymphs and females after oviposition, respectively. Transstadial transmission rates for nymphs exposed to virus as larvae, for adults exposed as larvae, and for adults exposed as nymphs were 33%, 11% and 46%, respectively. No evidence of transovarial transmission was obtained. Ticks in the stages of nymphs and adults were able to transmit the infection to uninfected hosts. This study demonstrated that H. marginatum could be involved in the natural circulation of WNV in Portugal.     

Transmission of Venezuelan equine encephalomyelitis virus by strains of Aedes albopictus (Diptera: Culicidae) collected in North and South America

Experimental studies were undertaken to ascertain the vector potential of North American (Houston and Alsace) and South American (Sao Paulo and Santa Teresa) strains of Aedes albopictus (Skuse) for an epizootic (Trinidad donkey) strain of Venezuelan equine encephalomyelitis (VEE) virus. Infection rates were similar in all four strains of Ae. albopictus tested after ingestion of VEE virus from a viremic hamster. Virus disseminated from the midgut to the hemocoel in about 80% of infected mosquitoes, regardless of the dose ingested (10(4.6) to 10(5.7) plaque-forming units per mosquito) or the time of extrinsic incubation (7-35 d). Although all four strains of this mosquito transmitted VEE virus by bite to hamsters, transmission rates were significantly higher for the South American strains (24%, 40 of 170) than for the North American strains (5%, 9 of 165). Although VEE virus has never been isolated from Ae. albopictus, the introduction of this species into the Americas may allow it to serve as an amplification vector in areas where epizootic strains of VEE are found or introduced.     

OspA antibodies inhibit the acquisition of Borrelia burgdorferi by Ixodes ticks.

Ixodes ticks are infected by Borrelia burgdorferi when larvae feed on spirochete-infected mice. We studied the acquisition of B. burgdorferi by larval ticks, characterized the production of outer surface protein A (OspA) by spirochetes entering larvae, and examined the effects of OspA antibodies on the establishment of B. burgdorferi infections in ticks. Most larvae were infected by spirochetes 24 to 48 h after placement on mice. OspA antibodies stained the first spirochetes observed in larvae, suggesting that OspA is synthesized early during the colonization of the vector. When OspA antibodies were administered to B. burgdorferi-infected mice and larvae were then placed on the animals, the severity of larval infection and the number of infected ticks (7 of 16) were decreased compared with that of controls (15 of 16). The inhibitory effects of OspA antibodies were observed with passive antibody transfer as well as active host-generated immunity. The lower larval infection rate observed in the presence of OspA antibodies was exacerbated after the larval molt since only 1 of 12 nymphs was infected, and none of the mice that were fed upon by these nymphs became infected with B. burgdorferi. Therefore, an OspA antibody response in mice altered the reservoir competence of the vertebrate host by inhibiting the movement of B. burgdorferi from the host to the vector.     

Transmission of Venezuelan equine encephalomyelitis virus by Aedes sollicitans and Aedes taeniorhynchus (Diptera: Culicidae).

Experimental studies compared the vector competence of Aedes sollicitans (Skuse) and Ae. taeniorhynchus (Wiedemann) collected on Assateague Island, Va., for an epizootic strain (Trinidad donkey) of Venezuelan equine encephalomyelitis (VEE) virus. Infection rates were significantly higher in Ae. sollicitans (101/107, 94%) than in Ae. taeniorhynchus (103/175, 59%), even though both species fed concurrently on the same infected hamsters. Similarly, dissemination and transmission rates were significantly higher in the Ae. sollicitans population tested. Although both Ae. taeniorhynchus and Ae. sollicitans are natural vectors of VEE virus, the latter species should be considered a more efficient vector of VEE epizootic strains, based on its greater susceptibility to infection and higher transmission rates.     

Vector competence of Mexican and Honduran mosquitoes (Diptera: Culicidae) for enzootic (IE) and epizootic (IC) strains of Venezuelan equine encephalomyelitis virus

Experimental studies evaluated the vector competence of Ochlerotatus taeniorhynchus (Wiedemann), Culex cancer Theobald, Culex pseudes (Dyar and Knab), Culex taeniopus Dyar and Knab, and a Culex (Culex) species, probably Culex quinquefasciatus Say, and Culex nigripalpus Theobald from Chiapas, Mexico, and Tocoa, Honduras, for epizootic (IC) and enzootic (IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. Culex pseudes was highly susceptible to infection with both the IC and IE strains of VEE (infection rates >78%). Patterns of susceptibility to VEE were similar for Oc. taeniorhynchus collected in Mexico and Honduras. Although Oc. taeniorhynchus was highly susceptible to the epizootic IC strains (infection rates > or = 95%, n = 190), this species was less susceptible to the enzootic IE strain (infection rates < or = 35%, n = 233). The Culex (Culex) species were refractory to both subtypes of VEE, and none of 166 contained evidence of a disseminated infection. Virus-exposed Cx. pseudes that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE. Although Oc. taeniorhynchus that fed on hamsters infected with the epizootic IC strain transmitted VEE efficiently, only one of six of those with a disseminated infection with the enzootic IE virus that fed on hamsters transmitted virus by bite. These data indicate that Cx. pseudes is an efficient laboratory vector of both epizootic and enzootic strains of VEE and that Oc. taeniorhynchus could be an important vector of epizootic subtypes of VEE.     

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.     

Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus.

An important question pertaining to the natural history of Venezuelan equine encephalitis (VEE) virus concerns the source of epizootic, equine-virulent strains. An endemic source of epizootic virus has not been identified, despite intensive surveillance. One of the theories of epizootic strain origin is that epizootic VEE viruses evolve from enzootic strains. Likely enzootic sources of VEE virus occur in Colombia and Venezuela where many of the epizootic outbreaks of VEE have occurred. We have determined the nucleotide sequences of the entire genomes of epizootic VEE subtype I-C virus, strain P676, isolated in Venezuela, and of enzootic VEE subtype I-D virus, strain 3880, isolated in Panama. VEE subtype I-D viruses are maintained in enzootic foci in Panama, Colombia, and Venezuela. The genomes of P676 and 3880 viruses differ from that of VEE subtype I-AB virus, strain Trinidad donkey (TRD), by 417 (3.6%) and 619 (5.4%) nucleotides, respectively. The translated regions of P676 and 3880 genomes differ from those of TRD virus by 54 (1.4%) and 66 (1.8%) amino acids, respectively. This study and the oligonucleotide fingerprint analyses of South American I-C and I-D viruses (Rico-Hesse, Roehrig, Trent, and Dickerman, 1988, Am. J. Trop. Med. Hyg. 38, 187-194) provide the most conclusive evidence to date suggesting that equine-virulent strains of VEE virus arise naturally from minor variants present in populations of I-D VEE virus maintained in enzootic foci in northern South America.     

Feeding efficiency of larval Hyalomma truncatum (Acari: Ixodidae) on hosts previously exposed to ticks.

The effect of guinea pig response to feeding larval Hyalomma truncatum Koch ticks was studied by measuring the percentage of ticks engorging and molting, their engorged weight, and hemoglobin content. Four guinea pigs were infested with 100, 200, 400, and 1,000 larvae, respectively, on three occasions at 21-d intervals, followed by a fourth infestation with 500 larvae. During the second, third, and fourth infestations, significantly fewer ticks engorged on each guinea pig than during the first infestation. The greatest reduction in percentage molting occurred during the fourth infestation on the animal with successive exposure to 400 larvae; only 24% of the ticks that fed molted. Ticks with the lowest mean weight and hemoglobin content also engorged on this animal during the fourth infestation. Guinea pigs exposed to 200 and 400 H. truncatum larvae elicited the greatest change in feeding efficiency during the fourth infestation. However, these hosts had no effect on a single subsequent fifth infestation with Amblyomma cajennense (F.) nymphs, as greater than 95% successfully engorged.     

Larval behavioral diapause regulates life cycle of Amblyomma cajennense (Acari: Ixodidae) in Southeast Brazil

Previous studies have determined that Amblyomma cajennense (F.) produces one generation per year in Southeastern Brazil. Larvae predominate from April to July, nymphs from July to October, and adults from October to March. In the current study, we observed engorged females, their eggs, and respective larvae in the field. Engorged females were released in grass plots under natural conditions, at monthly intervals, from October 2000 to March 2001. Preoviposition periods lasted 1-2 wk, and mean egg incubation periods lasted 5-7 wk. Consequently, larvae hatched at different periods from early December 2000 to early April 2001. However, all hatched larvae stayed confined to the plot ground, under the vegetation, until late April or early May 2001, when some larvae were seen questing on top of vegetation for the first time. Thereafter, larvae were always seen on this questing position until August 2001, when all larvae had died on the plots. In addition, premolt period data of engorged larvae and nymphs held in laboratory at different temperatures were used to construct a premolt mathematical model, which was applied to reported seasonal dynamic data of A. cajennense in the field. The premolt model indicated that the field intervals between larvae and nymphs and between nymphs and adults are controlled solely by nondiapause events, such as ground temperature. We concluded that the 1-yr generation pattern of A. cajennense in Southeastern Brazil is primarily controlled by larval behavioral diapause, shown by larvae born during spring/summer months, before their activity period at next autumn.     

Lyme disease in California: interrelationship of Ixodes pacificus (Acari: Ixodidae), the western fence lizard (Sceloporus occidentalis), and Borrelia burgdorferi

The relationship of immature western black-legged ticks, Ixodes pacificus Cooley and Kohls, to the western fence lizard, Sceloporus occidentalis Baird and Girard, and to the Lyme disease spirochete, Borrelia burgdorferi, was investigated in chaparral and woodland-grass habitats in northern California from 1984 to 1986. Immature ticks were found on lizards in spring and summer, but the prevalence and abundance of ticks on this host were considerably greater in spring. The peak of larval abundance preceded that of nymphs by several weeks, but there was considerable seasonal overlap between these parasitic stages. Larvae and nymphs attached primarily to the lateral nuchal pockets of lizards in chaparral (99.5%) and woodland-grass (91.8%). The numbers of larvae infesting lizards in spring fit the negative binomial distribution in woodland-grass but not in chaparral; insufficient data precluded similar analyses for nymphs. Tick loads did not differ significantly with respect to age or gender of the lizard. Spirochetal infection rates (range, 0-3.7%) in I. pacificus immatures were comparable in both habitats and were similar to those reported previously for adults of this tick. Overall, 1 (0.9%) of 117 larvae and 10 (1.8%) of 552 nymphs were infected with spirochetes resembling B. burgdorferi. Spirochetes were not observed in blood smears prepared from 261 wild-caught lizards, including five lizards fed upon by infected ticks at the time of collection. These and other findings suggest that S. occidentalis, although an important host of I. pacificus immatures, may be less important as a source for infecting ticks with B. burgdorferi.     

Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils

In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence.     

Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO2 tick trap

This study evaluates the impact of cattle-raising on ticks associated with wildlife in the Pantanal region of Brazil, by trapping free-living ticks using a CO(2) tick trap. The traps were equally distributed in two areas: a 600-ha protected area (PA) and the Nhumirim Ranch (NR). Both areas are contiguous and located at the sub-region of Nhecolandia (18 degrees 59'S; 56 degrees 39'W), Pantanal, Brazil. In each area, four habitat types were selected: cerrado (tropical savanna), forest patches, lakes and soda lakes. The last two are temporary lagoons of fresh and salted water, respectively. A total of 980 nymphs, 613 adults and 13 larvae of ticks Amblyomma were collected in 256 h of collection. In a pen of calves infested by Ornithodoros rostratus 114 specimens were collected, including larvae, nymphs and adults. Adults of A. cajennense and A. parvum were more abundant in the savanna at NR, but not differed among habitat types at PA. There was a tendency of having more nymphs in NR than in PA. The higher number of ticks found in the NR in comparison to the PA is not due to the presence of cattle itself but probably due to introduced dogs and horses, habitat alteration.     

Acquisition and Transmission of the Agent of Human Granulocytic Ehrlichiosis by Ixodes scapularis Ticks

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with Ixodes scapularis ticks. The median infectious dose for C3H mice was 10⁴ to 10⁵ organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.     

Transstadial transfer of West Nile virus by three species of ixodid ticks (Acari: Ixodidae)

Larvae and/or nymphs of four species of ixodid ticks, Ixodes scapularis Say, Amblyomma americanum (L.), Dermacentor andersoni Stiles, and Dermacentor variabilis Say, were fed to completion on laboratory hamsters or mice which had been inoculated with a West Nile (WN) virus isolate from Culex pipiens L. captured in Connecticut USA. Maximum titers in mice and hamsters were approximately 5 and two logs, respectively, lower than recorded (10 logs) in a naturally infected American crow, Corvus brachyrhynchos Brehm. WN virus was isolated in Vero cell culture from ticks and detected by TaqMan RT-polymerase chain reaction (PCR) in ticks that had completed their feeding as larvae or nymphs, and in I. scapularis, D. andersoni, and D. variabilis that had molted into the next stage of development. Naive hosts, fed upon by nymphs that as larvae had fed on viremic hosts, did not become infected. WN virus was isolated in Vero cell culture from one female I. scapularis and was detected by TaqMan RT-PCR in 24 adult I. scapularis, one D. andersoni, and two D. variabilis adults that had fed to completion as larvae on viremic hosts and as nymphs on naive mice or hamsters. Three species of ixodid ticks acquired WN virus from viremic hosts and transstadially passed the virus, but vector competency was not demonstrated.     

Transmission of Kyasanur Forest disease virus by Rhipicephalus haemaphysaloides ticks.

Larvae of Rhipicephalus haemaphysaloides were infected with Kaysanur Forest disease (KFD) virus by feeding on viraemic rodents and reared into next generation larvae. Fed larvae, nymphs, unfed adults, fed adult males, and females after oviposition were found infected, while the larvae were found free from infection. Nymphs and adults transmitted the infection by bite to rodents and rabbits respectively. The virus was also passed through a second rodent-tick cycle. Adult ticks showed a titre of 3.1 to 4.5 dex mouse LD50/0.03 ml, and the virus was also detected 245 days after infection.     

Experimental Venezuelan Equine Encephalomyelitis Virus Infection of the Bovine

Two groups of four dairy cows (Bos taurus) were infected subcutaneously with the epizootic Venezuelan equine encephalomyelitis virus (VEE) strains MF-8 and San Pelayo, respectively. Animals experienced no clinical illness, but all developed significant neutropenia. Virus was recovered once each from the blood of three animals but did not exceed 10(2.2) SMICLD(50) (Suckling mouse intracerebral lethal dose(50))/ml. Specific neutralizing antibodies appeared in the serum of all animals, but there were no significant differences in titers against different naturally occurring VEE subtypes. Dairy cattle thus appear to play no role in virus transmission during VEE epizootics but may serve as retrospective immunological sentinels of virus activity.     

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.     

A cloned DNA probe identifies Cowdria ruminantium in Amblyomma variegatum ticks.

Heartwater, caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma, is a constraint to ruminant animal production in sub-Saharan Africa. This rickettsial disease could spread from endemically infected areas of sub-Saharan Africa and certain Caribbean islands to other countries, including the United States, in which Amblyomma ticks exist. To detect C. ruminantium in tick vectors and animals, we made DNA probes from C. ruminantium DNA isolated from endothelial cell cultures. Two clones were evaluated; pCS20 from Crystal Springs (Zimbabwe) strain DNA had a 1,306-bp insert, and pCR9 from Kiswani (Kenya) strain DNA had a 754-bp insert. Both DNA probes detected 1 ng of Crystal Springs DNA; however, the pCS20 probe had a 10-fold-greater ability to discriminate between C. ruminantium DNA and DNA from other organisms. Also, the pCS20 probe did not hybridize to 400 ng (highest amount tested) of DNA from bovine cells, 3 protozoa, 3 rickettsiae, and 12 bacteria. In all experiments, C. ruminantium DNA was detected in midguts from 99 of 160 Amblyomma variegatum nymphs infected as larvae and in midguts from 38 of 80 adult ticks infected as nymphs but not in midguts from control nymphs and adults. The presence of C. ruminantium in nymphs and adults was confirmed by transmission of heartwater to goats. The DNA sequences of both probes were determined; synthetic oligonucleotides from pCS20 are recommended as DNA probes for C. ruminantium.