Infection rate of Aedes aegypti mosquitoes with dengue virus depends on the interaction between temperature and mosquito genotype.

Dengue fever is the most prevalent arthropod-transmitted viral disease worldwide, with endemic transmission restricted to tropical and subtropical regions of different temperature profiles. Temperature is epidemiologically relevant because it affects dengue infection rates in Aedes aegypti mosquitoes, the major vector of the dengue virus (DENV). Aedes aegypti populations are also known to vary in competence for different DENV genotypes. We assessed the effects of mosquito and virus genotype on DENV infection in the context of temperature by challenging Ae. aegypti from two locations in Vietnam, which differ in temperature regimes, with two isolates of DENV-2 collected from the same two localities, followed by incubation at 25, 27 or 32°C for 10 days. Genotyping of the mosquito populations and virus isolates confirmed that each group was genetically distinct. Extrinsic incubation temperature (EIT) and DENV-2 genotype had a direct effect on the infection rate, consistent with previous studies. However, our results show that the EIT impacts the infection rate differently in each mosquito population, indicating a genotype by environment interaction. These results suggest that the magnitude of DENV epidemics may not only depend on the virus and mosquito genotypes present, but also on how they interact with local temperature. This information should be considered when estimating vector competence of local and introduced mosquito populations during disease risk evaluation.

Saliva collection via capillary method may underestimate arboviral transmission by mosquitoes.

BACKGROUND: Arthropod-borne viruses (arboviruses) impose a major health and economic burden on human populations globally, with mosquitoes serving as important vectors. Measuring the ability of a mosquito population to transmit an arbovirus is important in terms of evaluating its public health risk. In the laboratory, a variety of methods are used to estimate arboviral transmission by mosquitoes, including indirect methods involving viral detection from mosquito saliva collected by forced salivation. The accuracy of indirect methods to estimate arbovirus transmission to live animal hosts has not been fully evaluated. METHODS: We compared three commonly used proxies of arboviral transmission, namely, the presence of virus in mosquito legs, in salivary glands (SG) and in saliva collected in capillary tubes using forced salivation, with direct transmission estimates from mosquitoes to suckling mice. We analyzed five vector-virus combinations, including Aedes aegypti infected with chikungunya virus, West Nile virus and Zika virus; Culex quinquefasciatus infected with West Nile virus; and Aedes triseriatus infected with La Crosse virus. RESULTS: Comparatively, the methods of detecting virus infection in mosquito legs and in SG were equally accurate in predicting transmission. Overall, the presence of virus in mosquito legs was a more accurate predictor of transmission than the commonly implemented viral detection method using forced salivation into a capillary tube, and was subject to less technical variation. CONCLUSIONS: These results suggest that, in general, forced salivation methods tend to underestimate virus transmission, and they provide confidence in the use of mosquito leg screens to evaluate the transmission potential of a mosquito population.

Identification of bloodmeals in Anopheles quadrimaculatus and Anopheles punctipennis from eastern equine encephalitis virus foci in northeastern U.S.A.

The host-feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)-based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian-derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White-tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8-97.7% of all bloodmeals identified. The predominance of white-tailed deer as a source of bloodmeals supports enzootic amplification of deer-associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse- and two human-derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian-derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp-shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic-epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.

Diversity of Babesia infecting deer ticks (Ixodes dammini).

To determine whether the presence of nonpathogenic piroplasms may confound field estimates of risk of Babesia microti infection, we identified sporozoites infecting the salivary glands of deer ticks (Ixodes dammini) by parallel microscopy and polymerase chain reaction assays. Piroplasms were evident in 14.4% of adult ticks from sites in the northcentral and northeastern United States. Of these, 83.3% contained DNA characteristic of Ba. odocoilei. This cervid piroplasm was detected in all of the sites examined and generally was more prevalent than was Ba. microti. Because deer ticks transmit both Ba. odocoilei and Ba. microti, estimates of pathogen prevalence based solely on microscopy may overestimate the risk of human babesiosis.

A subtropical case of human babesiosis.

This report constitutes the first well-documented case of symptomatic human babesiosis from a subtropical site, south of the 40th parallel. This paper describes the definitive identification of Babesia divergens infection in a splenectomized patient from the Canary Islands.

Differential susceptibility of Aedes aegypti to infection by the American and Southeast Asian genotypes of dengue type 2 virus.

Outbreaks of dengue hemorrhagic fever have coincided with the introduction of the Southeast (SE) Asian genotype of dengue type 2 virus in the Western Hemisphere. This introduced genotype appears to be rapidly displacing the indigenous, American genotype of dengue 2 virus throughout the region. These field observations raise the possibility that the SE Asian genotype of dengue 2 is better adapted for vector transmission than its American counterpart. To evaluate this hypothesis, we compared the ability of viral strains of the SE Asian and American genotypes to infect, replicate, and disseminate within vector mosquitoes (Aedes aegypti). Viral strains of the SE Asian genotype tended to infect and disseminate more efficiently in mosquitoes than did variants of the American genotype. These differences, however, were observed solely in field-derived mosquitoes, whereas viral infection rates were virtually identical in the laboratory-adapted Rockefeller colony of Ae. aegypti. Our findings could provide a physiological basis for the contrasting patterns of dengue virus genotype transmission and spread. Such an understanding of functional differences between viral strains and genotypes may ultimately improve surveillance and intervention strategies.

Pyoderma gangrenosum. A diagnosis not to be missed.

We describe a case of pyoderma gangrenosum which presented with severe wound breakdown after elective hip replacement. The patient was treated successfully with minimal wound debridement and steroids. This diagnosis should always be considered when confronted with an enlarging painful skin lesion which does not grow organisms when cultured and fails to respond to antibiotic therapy, especially if there are similar lesions in other sites. In patients who have a past history of pyoderma gangrenosum, prophylactic steroids may be indicated at the time of surgery or may be required early in the postoperative period.

Signaled avoidance in the eye withdrawal reflex of the green crab.

Learning in a signaled avoidance procedure was studied in the eye withdrawal reflex of the green crab, Carcinus maenas. A puff of air to the eye, which causes eye retraction, was used as the unconditioned stimulus (US). A mild vibration on the carapace, which has no effect on untrained animals, was used as a warning (conditioned) stimulus (CS). Eye withdrawal during the CS led to the omission of the otherwise scheduled US. Acquisition was rapid, reaching about 75% avoidance after 30 trials. Extinction occurred slowly over the course of 40 CS-only trials. Yoked controls did not perform as well. The behavior of experimental animals in the avoidance procedure was found to be essentially identical to the performance of animals subjected to a classical conditioning paradigm in which CS responses had no effect on US presentation. Additional groups of animals were subjected to experiments in which (a) avoidance conditioning (60 trials) was followed by classical conditioning (40 trials) or (b) classical conditioning was followed by avoidance. The behavior of these groups was, again, essentially identical. The results suggest that there may be an underlying Pavlovian mechanism for the learned response, although the contribution of an operant process is not excluded. The results expand the range of invertebrate animals in which fundamental conditioning phenomena can be demonstrated, and may provide a neuronal model for learning in a signaled avoidance procedure.

Serum-plasma separator in amylase assay.

A modification of the Amylochrome method for amylase activity is described and evaluated. The modification consists of using Sure-Sep II Serum-Plasma Separators for the separation of the supernatant and precipitant in this procedure. Thirty patient sera and 20 control sera were run in duplicate with and without the separators. Comparison studies of the reference and separator methods show that the separator does not interfere with amylase activity, and there is excellent correlation (r = 0.99) between the methods. The time saved during centrifugation allows for easier and faster determination of amylase activity.

Risk of Lyme disease: perceptions of residents of a Lone Star tick-infested community.

BACKGROUND: Lone Star ticks (Amblyomma americanum) have been suggested as a vector of the agent of Lyme disease (Borrelia burgdorferi sensu lato) in the USA, based on associations with an infection manifesting mainly as erythema migrans. In laboratory experiments, however, they failed to transmit B. burgdorferi sensu stricto. METHODS: In this study, carried out from 1994 to 1996, we determined the seroprevalences of B. burgdorferi (1.2%), Ehrlichia chaffeensis (7%), E. phagocytophila (0%), Rickettsia rickettsii (0%), R. typhi (0%), Coxiella burneti (0%), Francisella tularensis (0%), and Babesia microti (0%) by standard serological methods for 325 residents (97% of the total population) of Gibson Island, coastal Maryland, USA, where 15% of the residents reported having had Lyme disease within a recent 5-year span. FINDINGS: Of the 167 seronegative individuals who were followed up prospectively for 235 person-years of observation, only 2 (0.85%) seroconverted for B. burgdorferi. Of 1556 ticks submitted from residents, 95% were identified as Lone Star ticks; only 3% were deer ticks (Ixodes dammini), the main American vector of Lyme disease. B. burgdorferi s.s. infected 20% of host-seeking immature deer ticks, and borreliae ("B. lonestari") were detected in 1-2% of Lone Star ticks. Erythema migrans was noted in 65% of self-reports of Lyme disease, but many such reports indicated that the rash was present while the tick was still attached, suggesting a reaction to the bite itself rather than true Lyme disease. Sera from individuals reporting Lyme disease generally failed to react to B. burgdorferi or any other pathogen antigens. CONCLUSION: The residents of Gibson Island had an exaggerated perception of the risk of Lyme disease because they were intensely infested with an aggressively human-biting and irritating nonvector tick. In addition, a Lyme disease mimic of undescribed etiology (named Masters' disease) seems to be associated with Lone Star ticks, and may confound Lyme disease surveillance. The epidemiological and entomological approach used in this study might fruitfully be applied wherever newly emergent tickborne zoonoses have been discovered.

Lone star tick-infecting borreliae are most closely related to the agent of bovine borreliosis.

Although Borrelia theileri, the agent of bovine borreliosis, was described at the turn of the century (in 1903), its relationship with borreliae causing Lyme disease or relapsing fever remains undescribed. We tested the previously published hypothesis that spirochetes infecting Lone Star ticks (Amblyomma americanum) may comprise B. theileri by analyzing the 16S ribosomal DNAs (rDNAs) and flagellin genes of these spirochetes. B. theileri, the Amblyomma agent, and B. miyamotoi formed a natural group or clade distinct from but most closely related to that of the relapsing fever spirochetes. B. theileri and the Amblyomma agent were 97 and 98% similar at the nucleotide level within the analyzed portions of the 16S rDNA and the flagellin gene respectively, suggesting a recent divergence. The agent of bovine borreliosis might be explored as a surrogate antigen for the as-yet-uncultivatable Amblyomma agent in studies designed to explore the etiology of a Lyme disease-like infection associated with Lone Star ticks.

Duration of tick attachment required for transmission of granulocytic ehrlichiosis.

Deer tick-transmitted pathogens such as Lyme disease spirochetes and babesiae appear to require a period of reactivation and replication during the tick's blood meal before it is able to infect a host. The duration of nymphal tick attachment that is required for transmission of the agent of human granulocytic ehrlichiosis (HGE) was determined by removing feeding ticks from mice at various time points. As with spirochetes and babesiae, ehrlichiae infected few mice when ticks were removed prior to 36 h of tick attachment. This "grace period" may serve as a modifying factor in the epidemiology of this newly emergent zoonosis and help physicians make informed decisions concerning management of tick bites in HGE-endemic areas.

A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini.

To determine if eastern North American Ixodes dammini, like related ticks in Eurasia, maintain tick-borne encephalitis group viruses, we analyzed ticks collected from sites where the agent of Lyme disease is zoonotic. Two viral isolates were obtained by inoculating mice with homogenates from tick salivary glands. The virus, which was described by reverse transcriptase polymerase chain reaction and direct sequencing of the amplification products, was similar to, but distinct from, Powassan virus and is provisionally named "deer tick virus." Enzootic tick-borne encephalitis group viruses accompany the agents of Lyme disease, babesiosis, and granulocytic ehrlichiosis in a Holarctic assemblage of emergent deer tick pathogens.