Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay.

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Modulation of host immunity by haematophagous arthropods.

The medical and veterinary public-health importance of haematophagous arthropods is immense and continuing to increase because of the emergence of new vector-borne infectious agents and the resurgence of well known ones. Control of blood-feeding arthropods and the pathogens they transmit is compounded by drug, insecticide and acaricide resistance. Novel control strategies are needed. Immunological control is one very promising approach to these problems. In order to develop anti-arthropod vaccines that block pathogen transmission and establishment, the immunological interactions occurring at the interface of the blood-feeding arthropod and host must be characterized. An important component of these interactions is arthropod modulation of the host's innate and acquired, specific immune defences. This review discusses current knowledge regarding the ability of haematophagous arthropods to alter their hosts' immune defences, the impact of those changes on pathogen transmission, the molecular bases for the immunomodulation, and strategies for identification of the molecules in arthropod saliva that are responsible for the immunomodulation.

Influence of repeated infestations with pathogen-free Ixodes scapularis (Acari: Ixodidae) on in vitro lymphocyte proliferation responses of C3H/HeN mice.

In the United States, Ixodes scapularis Say has been implicated as the vector of at least three human pathogens. Tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on in vitro proliferative responses of splenic lymphocytes from C3H/HeN mice. Lymphocytes from repeatedly infested and uninfested mice were exposed to concanavalin A (Con A), Escherichia coli Castellini & Chalmers lipopolysaccharide (LPS), or I. scapularis salivary gland soluble proteins (SGSP), to determine if lymphocyte responses differed between tick-exposed and nonexposed mice. Female C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-d tick-free period between each exposure. After each infestation, tick biology parameters were measured and lymphocyte proliferative responses assessed. Acquired resistance to I. scapularis was not evident in mice subjected to tick feeding. Significant differences in the responses of lymphocytes exposed to I. scapularis SGSP were observed between infested and noninfested mice. In contrast, few differences between infested and noninfested mice were evident for lymphocytes exposed to Con A or LPS. Our results suggest that repeated exposure to I. scapularis nymphs does not affect Con A or LPS-induced proliferation of splenic lymphocytes, but significantly effects lymphocyte responses to tick salivary gland antigens.

Influence of soluble proteins from the salivary glands of ixodid ticks on the in-vitro proliferative responses of lymphocytes from BALB/c and C3H/HeN mice.

In the U.S.A., Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans by the ticks Ixodes scapularis and I. pacificus. Tick modulation of host immunity is an important factor in tick transmission of such pathogens. The proliferative responses of lymphocytes from BALB/c and C3H/HeN mice exposed to the salivary-gland soluble proteins (SGSP) of I. scapularis, I. pacificus or Dermacentor andersoni were therefore compared in vitro. This produced the present report, the first to describe the effects of I. pacificus SGSP on the proliferative responses of a host's lymphocytes in vitro. The effects of four concentrations of SGSP from each tick species were evaluated with unstimulated, and concanavalin-A-stimulated lymphocytes of each mouse strain. The responses of lymphocytes from both mouse strains were significantly effected when exposed to SGSP derived from each tick species. Responses of the unstimulated lymphocytes to SGSP indicated that the proteins from I. pacificus suppressed in-vitro lymphocyte proliferation to a greater degree than those from the other species investigated. For the concanavalin-A stimulated cells, however, suppression of the proliferative responses was greatest for cells exposed to I. scapularis SGSP.

Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi.

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.

Decorin-binding protein A (DbpA) of Borrelia burgdorferi is not protective when immunized mice are challenged via tick infestation and correlates with the lack of DbpA expression by B. burgdorferi in ticks.

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi-infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.

Cytokine responses of C3H/HeN mice infested with Ixodes scapularis or Ixodes pacificus nymphs.

Lyme borreliosis, caused by Borrelia burgdorferi, is transmitted by Ixodes scapularis in the eastern and midwestern United States and by Ixodes pacificus in the far-Western United States. Studies have shown that infestation with I. scapularis nymphs modulates host cytokine production; however, the influence of I. pacificus infestation on host cytokines remains uninvestigated. This study demonstrated how repeated infestations with pathogen-free I. scapularis or I. pacificus nymphs affects the production of the macrophage cytokines IL-1beta, IL-6 and tumour necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma by C3H/HeN mice. Female mice were infested once or twice with pathogen-free I. scapularis or I. pacificus nymphs, with a 14-day tick-free period between exposures. After each infestation, tick biology parameters were assessed and macrophage and T lymphocyte cytokine production measured by antigen capture ELISA. Acquired resistance to tick feeding did not develop after infestation with either tick species. Differences in cytokine production were observed between infested and noninfested mice, and between mice infested with either I. scapularis or I. pacificus nymphs. Infestations polarized cytokine production towards a Th2 cytokine profile, with suppression of pro-inflammatory Th1 cytokines. This pattern of cytokine production is more pronounced for I. pacificus infested mice.

Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.

We previously reported on the existence of a family of lipoprotein genes, designated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297. A distinguishing feature of the 2.9 lipoproteins were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the mlp genes were expressed when B. burgdorferi was cultivated in vitro at 34 degrees C, although mlp-9 and mlp-10 transcripts were expressed at very low levels. Additional combined immunoblotting and comparative reverse transcription-PCR analyses performed on borreliae cultivated in vitro at 23, 34, or 37 degrees C indicated that although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the mlp genes were upregulated during B. burgdorferi replication at 37 degrees C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growth of B. burgdorferi in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with Ixodes scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulated by temperature (37 degrees C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease.

Ixodes scapularis: effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice.

Schoeler, G. B., Manweiler, S. A., and Wikel, S. K. 1999. Ixodes scapularis: Effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice. Experimental Parasitology 92, 239-248. Ixodes scapularis is the principal vector in the United States of Borrelia burgdorferi, the causative agent of Lyme borreliosis, the human granulocytic ehrichiosis agent, and Babesia microti. Infestation with I. scapularis nymphs has previously been shown to modulate host T lymphocyte cytokine production. Tick-induced host immunomodulation is increasingly recognized as a contributing factor in successful transmission and/or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on the production of the macrophage cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-10, and interferon-gamma in both BALB/c and C3H/HeN mice. The pattern of T lymphocyte cytokine production was evaluated to determine if repeated tick infestation polarizes the immune response toward a Th-1 or Th-2 cytokine profile. Female BALB/c and C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-day tick-free period between each exposure. After each infestation, tick biology parameters were measured and macrophage and T lymphocyte cytokine production was assessed. Elaboration of T lymphocyte and macrophage cytokines was quantitated by antigen capture enzyme-linked immunosorbent assay. Acquired resistance to I. scapularis feeding was not developed by either mouse strain. Significant differences in cytokine production were observed between infested and noninfested mice, as well as between the two mouse strains, following tick infestation. Infestation of both strains with pathogen-free I. scapularis results in a polarization of the host immune response toward a Th-2, anti-inflammatory pattern, with a corresponding suppression of Th-1 responses.

Diel activity of nymphal Dermacentor occidentalis and Ixodes pacificus (Acari: Ixodidae) in relation to meteorological factors and host activity periods.

Relation of diel activity and questing behavior of nymphal Dermacentor occidentalis Marx and Ixodes pacificus Cooley & Kohls to meteorological factors was investigated in a shaded versus a sun-exposed outdoor arena. Oak-woodland soil covered partially with leaf litter and small rocks, and 24 vertically oriented grass stems 2.5, 5.0, 10.0, and 20.0 cm tall were provided as substrate and potential questing sites. Tick activity and weather conditions were monitored bihourly during 15 diel (24-h) experiments (D. occidentalis, 8; I. pacificus, 7). In shade, D. occidentalis was active throughout the day, but questing occurred mainly at night and in the morning on grass stems or atop soil when temperatures were cool and relative humidities high. Ticks seemed to prefer to quest at heights between approximately 4 and 10 cm. The time of day and height at which D. occidentalis quested on grass stems coincided with the activity periods and size of its lagomorph and rodent hosts. Low percentages (< or = 15%) of I. pacificus nymphs (n = 100 or 200) were active atop soil or leaf litter at night or sporadically throughout the day, but none ascended grass stems. This finding was reconfirmed by monitoring diurnal behavior of nymphs in an outdoor aquarium having leaf litter as substrate; < or = 4% of 53 ticks were detected on the topmost layer of leaves and, of those, most I. pacificus were situated on the lower versus the upper surfaces of such leaves. Activity of I. pacificus was correlated positively with relative humidity and negatively with soil temperature in one experiment. In the sun-exposed arena, ticks of both species died within 9-11 d as daytime soil-surface temperatures sometimes reached maximums of 73-77 degrees C and relative humidities dropped to 14-24%. In contrast, D. occidentalis and I. pacificus survived for up to 6 and 8 wk, respectively, in the shaded arena. After its introduction into the shaded arena, the western fence lizard (Sceloporus occidentalis Baird & Girard) acquired more I. pacificus nocturnally while asleep in soil than during its diurnal period of activity above ground. Sleeping wild lizards also became infested more often and had significantly greater burdens of I. pacificus subadults, primarily larvae, than diurnally active lizards. Collectively, these findings demonstrate that I. pacificus subadults are capable of locating and attaching to their saurian hosts subterraneanly as well as above ground.

Efficiency of transovarial transmission of the Lyme disease spirochete, Borrelia burgdorferi, in the western blacklegged tick, Ixodes pacificus (Acari: Ixodidae).

The efficiency of transovarial transmission of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner was evaluated in Ixodes pacificus Cooley & Kohls collected from two areas of northern California where Lyme disease is endemic. In total, 132 (8.8%) of 1,499 replete females examined by direct immunofluorescence were demonstrated to be infected with B. burgdorferi. Larvae or eggs from 119 of these females were examined for the presence of spirochetes by direct immunofluorescence, placing them in culture, or both; none was found to contain B. burgdorferi. The fecundity of 20 midgut-infected (mean = 874.2) and 20 uninfected (mean = 1,048.3) I. pacificus females did not differ statistically. Likewise, the fertility of infected (mean = 87.0%) and uninfected (mean = 89.9%) females and the mean engorged weights of both groups (infected, 120.8 mg versus uninfected, 132.7 mg), were comparable. The fecundity, fertility, and mean weights of six replete females having ovarian infections, six females having midgut-restricted infections, and six uninfected females were also similar. We conclude that transovarial transmission is not efficient for maintaining B. burgdorferi in populations of I. pacificus, a known vector of that pathogen. Infection with the spirochete does not appear to affect either feeding or reproductive success adversely in females of this tick.