Acquisition of Borrelia burgdorferi Infection by Larval Ixodes scapularis (Acari: Ixodidae) Associated With Engorgement Measures.

Measuring rates of acquisition of the Lyme disease pathogen, Borrelia burgdorferi sensu lato Johnson, Schmid, Hyde, Steigerwalt & Brenner, by the larval stage of Ixodes scapularis Say is a useful tool for xenodiagnoses of B. burgdorferi in vertebrate hosts. In the nymphal and adult stages of I. scapularis, the duration of attachment to hosts has been shown to predict both body engorgement during blood feeding and the timing of infection with B. burgdorferi. However, these relationships have not been established for the larval stage of I. scapularis. We sought to establish the relationship between body size during engorgement of larval I. scapularis placed on B. burgdorferi-infected, white-footed mice (Peromyscus leucopus Rafinesque) and the presence or absence of infection in larvae sampled from hosts over time. Body size, time, and their interaction were the best predictors of larval infection with B. burgdorferi. We found that infected larvae showed significantly greater engorgement than uninfected larvae as early as 24 h after placement on a host. These findings may suggest that infection with B. burgdorferi affects the larval feeding process. Alternatively, larvae that engorge more rapidly on hosts may acquire infections faster. Knowledge of these relationships can be applied to improve effective xenodiagnosis of B. burgdorferi in white-footed mice. Further, these findings shed light on vector-pathogen-host interactions during an understudied part of the Lyme disease transmission cycle.

Relative humidity and activity patterns of Ixodes scapularis (Acari: Ixodidae).

Laboratory studies have shown clear relationships between relative humidity (RH) and the activity and survival of Ixodes scapularis Say (blacklegged tick). However, field studies have produced conflicting results. We examined this relationship using weekly tick count totals and hourly RH observations at three field sites, stratified by latitude, within the state of Rhode Island. Records of nymphal tick abundance were compared with several RH-related variables (e.g., RH at time of sampling and mean weekly daytime RH). In total, 825 nymphs were sampled in 2009, a year of greater precipitation, with a weighted average leaf litter RH recorded at time of sampling of 85.22%. Alternatively, 649 nymphs were collected in 2010, a year of relatively low precipitation, and a weighted average RH recorded at time of sampling was 75.51%. Negative binomial regression analysis of tick count totals identified cumulative hours < 82% RH threshold as a significant factor observed in both years (2009: P = 0.0037; 2010: P < 0.0001). Mean weekly daytime RH did not significantly predict tick activity in either year. However, mean weekly daytime RH recorded with 1-wk lag before sample date was a significant variable (P = 0.0016) in 2010. These results suggest a lag effect between moisture availability and patterns of tick activity and abundance. Differences in the relative importance of each RH variable between years may have been due to abnormally wet summer conditions in 2009.

Highly variable acquisition rates of Ixodes scapularis (Acari: Ixodidae) by birds on an Atlantic barrier island.

Acquisition of ticks by bird hosts is a central process in the transmission cycles of many tick-borne zoonoses, but tick recruitment by birds has received little direct study. We documented acquisition of Ixodes scapularis Say on birds at Fire Island, NY, by removing ticks from mist-netted birds, and recording the number of ticks on birds recaptured within 4 d of release. Eight bird species acquired at least 0.8 ticks bird(-1) day(-1) during the seasonal peak for at least one age class of I. scapularis. Gray Catbirds, Eastern Towhees, Common Yellowthroats, and Northern Waterthrushes collectively accounted for 83% of all tick acquisitions; and six individuals apportioned among Black-billed Cuckoo, Gray Catbird, Eastern Towhee, and Common Yellowthroat were simultaneously infested with both larvae and nymphs. Bird species with the highest acquisition rates were generally ground foragers, whereas birds that did not acquire ticks in our samples generally foraged above the ground. Tick acquisition by birds did not differ between deciduous and coniferous forests. Among the 15 bird species with the highest recruitment rates, acquisition of nymphs was not correlated with acquisition of larvae. Tick acquisition rates by individual bird species were not correlated with the reservoir competence of those species for Lyme borreliae. However, birds with high tick acquisition rates can contribute large numbers of infected ticks, and thus help maintain the enzootic cycle, even if their levels of reservoir competence are relatively low.

Comparative analysis of distribution and abundance of West Nile and eastern equine encephalomyelitis virus vectors in Suffolk County, New York, using human population density and land use/cover data.

Five years of CDC light trap data from Suffolk County, NY, were analyzed to compare the applicability of human population density (HPD) and land use/cover (LUC) classification systems to describe mosquito abundance and to determine whether certain mosquito species of medical importance tend to be more common in urban (defined by HPD) or residential (defined by LUC) areas. Eleven study sites were categorized as urban or rural using U.S. Census Bureau data and by LUC types using geographic information systems (GISs). Abundance and percent composition of nine mosquito taxa, all known or potential vectors of arboviruses, were analyzed to determine spatial patterns. By HPD definitions, three mosquito species, Aedes canadensis (Theobald), Coquillettidia perturbans (Walker), and Culiseta melanura (Coquillett), differed significantly between habitat types, with higher abundance and percent composition in rural areas. Abundance and percent composition of these three species also increased with freshwater wetland, natural vegetation areas, or a combination when using LUC definitions. Additionally, two species, Ae. canadensis and Cs. melanura, were negatively affected by increased residential area. One species, Aedes vexans (Meigen), had higher percent composition in urban areas. Two medically important taxa, Culex spp. and Aedes triseriatus (Say), were proportionally more prevalent in residential areas by LUC classification, as was Aedes trivittatus (Coquillett). Although HPD classification was readily available and had some predictive value, LUC classification resulted in higher spatial resolution and better ability to develop location specific predictive models.

Integrated pest management and allocation of control efforts for vector-borne diseases.

Applications of various control methods were evaluated to determine how to integrate methods so as to minimize the number of human cases of vector-borne diseases. These diseases can be controlled by lowering the number of vector-human contacts (e.g., by pesticide applications or use of repellents), or by lowering the proportion of vectors infected with pathogens (e.g., by lowering or vaccinating reservoir host populations). Control methods should be combined in such a way as to most efficiently lower the probability of human encounter with an infected vector. Simulations using a simple probabilistic model of pathogen transmission suggest that the most efficient way to integrate different control methods is to combine methods that have the same effect (e.g., combine treatments that lower the vector population; or combine treatments that lower pathogen prevalence in vectors). Combining techniques that have different effects (e.g., a technique that lowers vector populations with a technique that lowers pathogen prevalence in vectors) will be less efficient than combining two techniques that both lower vector populations or combining two techniques that both lower pathogen prevalence, costs being the same. Costs of alternative control methods generally differ, so the efficiency of various combinations at lowering human contact with infected vectors should be estimated at available funding levels. Data should be collected from initial trials to improve the effects of subsequent interventions on the number of human cases.

Extended lung expression and increased tissue localization of viral IL-10 with adenoviral gene therapy.

IL-10 is a pleiotropic cytokine that acts as an important regulator of macrophage, T cell, and natural killer cell functions. Human IL-10 (hIL-10) has both stimulatory and inhibitory effects on a wide variety of cell types. Viral IL-10 (vIL-10) possesses only a subset of hIL-10's activities, predominantly its suppression of cytokine synthesis by T helper type 1 clones. In the present report, we evaluated tissue accumulation and biological activity of hIL-10 and vIL-10 in vivo in individual organs by using a first-generation adenoviral (Ad) vector administered intratracheally and intravenously. We report the observation that Ad vectors delivering vIL-10, but not hIL-10, are associated with prolonged expression in the lung (>42 days) when delivered intratracheally. In contrast, there was no prolongation in vIL-10 expression when Ad vectors were intravenously administered, although vIL-10 levels in the tissue, but not serum, were markedly increased relative to hIL-10. Moreover, we report an augmented capacity of expressed vIL-10 versus hIL-10 to suppress the acute inflammatory responses in the lung to intratracheal administration of Ad. These findings confirm fundamental differences in Ad-induced expression of vIL-10 and hIL-10 when administered to the lungs. The results further suggest that Ad vectors expressing vIL-10 may have a role as anti-inflammatory agents in the treatment of acute and chronic lung inflammation.

Pathogenicity of Bacillus thuringiensis variety kurstaki to Ixodes scapularis (Acari: Ixodidae).

Pathogenicity of the entomopathogenic bacterium Bacillus thuringiensis variety kurstaki de Barjac & Lemille was tested against the black-legged tick, Ixodes scapularis Say. Engorged larvae dipped in a solution of 10(8) spores per milliliter showed 96% mortality 3 wk after infection. The LC50 value for engorged larve (concentration required to kill 50% of ticks) was 10(7) spores per milliliter. B. thuringiensis shows considerable potential as a microbial control agent for the management of I. scapularis.

The life and times of adenoviruses.

With Wallace Rowe et al.'s and Hilleman and Werner's isolations of viruses, subsequently termed "adenoviruses," a new area of research opened for me and gradually for many others. I was quickly able to associate the viruses with diseases in humans, and then our attention turned to the structure of the virion and how it replicated. Many virologists entered these areas of adenovirus research, for they were the central themes for most virologists at that time. We obtained more and more knowledge of the structure of the virion, its genome, and how it replicated and killed cells in culture so that they could no longer divide, although the virus infection did not lyse the infected cells, but we did not have the slightest idea how Ad5 produced disease in vivo. Then Wallace Clyde's timely note appeared, and we entered an exciting and profitable new field: an investigation of the mechanism by which Ad5 produces pneumonia. It must again be emphasized that the pneumonia that WtAd5 produces in cotton rats is pathologically very similar to that induced in humans. One of our earliest sets of experiments in the cotton rats was designed to determine whether region E3 was really nonessential even though the genes contained therein were not required for viral replication. We soon demonstrated that deletion of the E3 region produced a mutant that induced a highly pathogenic viral pneumonia. The potential role in pathogenesis of each of the genes within the E3 region was then investigated. Of maximum importance was the finding that deletion of the 19-kDa gene near the 5' end of the region produced a severe inflammatory response. This result led to the discovery that the E3 19-kDa protein regulated expression of the MHC factor on the surface of infected cells, and deletion of this gene produced a marked increase in MHC on the surfaces of infected cells and, therefore, a marked increase in the response of cytotoxic T cells. In addition, deletion of the gene encoding the 14.7-kDa protein, which was situated at the 3' end of the E3 region, resulted in an increase in polymorphonuclear leukocytes in the inflammatory response. A number of these findings led to hypotheses that could not be tested in the cotton rat since the necessary reagents were not available. Fortunately, our findings that only early viral genes are required to produce full pathogenesis led us to test mice because we had shown in a culture of mouse cells that all of the early viral genes are expressed. The C57BL/6N mouse proved to be an excellent host in which Ad5 produced full pulmonary inflammation. Thus, it was possible to test our hypotheses and to demonstrate their validity, showing that the virus induces cytokine elaboration, as well as to demonstrate the role of cytotoxic T cells in permitting Ad5 to produce persistent infections in lymphoid cells of organs such as the adenoid, from which the first adenovirus was isolated, and which had immediately led to my interest in investigating it and helping to develop the story of adenoviruses.

Preliminary survey for entomopathogenic fungi associated with Ixodes scapularis (Acari: Ixodidae) in southern New York and New England, USA.

Free-living larval, nymphal, and adult Ixodes scapularis Say were collected from scattered locales in southern New England and New York to determine infection rates with entomopathogenic fungi. Infection rates of larvae, nymphs, males, and females were 0% (571), 0% (272), 0% (57), and 4.3% (47), respectively. Two entomopathogenic fungi were isolated from field-collected I. scapularis females from Fire Island, NY. Isolates were identified as Verticillium lecanii (Zimmermann) Viegas and Verticillium sp. (a member of the Verticillium lecanii species complex).

Role of the type 5 adenovirus gene encoding the early region 1B 55-kDa protein in pulmonary pathogenesis.

Comparison of the inflammatory response of Sigmodon hispidus cotton rats to pulmonary infection with wild-type 5 adenovirus (Ad5) or with a viral mutant, in which the early region 1B gene encoding a 55-kDa protein, Ad5dl110 (dl110), was deleted, indicated that the inflammation in animals infected with dl110 was markedly reduced compared with the inflammation in animals infected with wild-type Ad5, although both viruses replicated to the same extent. Comparable experiments done with C57BL/6 mice yielded identical results, even though only the early phase of gene expression essential for viral replication occurs in mice. Cytokine analysis of infected mouse lungs indicated that tumor necrosis factor-alpha and IL-6 were produced in relatively large quantities in wild-type Ad5-infected mice and at significantly lower levels in dl110-infected mice during the early stages of infection.

Influence of deer abundance on the abundance of questing adult Ixodes scapularis (Acari: Ixodidae).

Nymphal and adult Ixodes scapularis Say were sampled by flagging at 2 sites on a barrier island, Fire Island, NY, and at 2 sites on the nearby mainland. Nymphal densities did not differ consistently between island and mainland sites, but adult densities were consistently lower on the island. We tested whether lower adult densities on the island resulted from greater nymphal mortality on the island than the mainland, or whether adult ticks on the island were poorly sampled by flagging because they had attached abundantly to deer, which were common on Fire Island. Differential nymphal mortality on islands versus mainland did not explain this difference in adult densities because survival of flat and engorged nymphs in enclosures was the same at island and mainland sites. Ticks were infected by parasitic wasps on the island and not the mainland, but the infection rate (4.3%) was too low to explain the difference in adult tick densities. In contrast, exclusion of deer by game fencing on Fire Island resulted in markedly increased numbers of adult ticks in flagging samples inside compared with samples taken outside the exclosures. Therefore, the scarcity of adult ticks in flagging samples on Fire Island resulted, at least in part, from the ticks being unavailable to flagging samples because they were on deer hosts. Differences in the densities of flagged ticks inside and outside the exclosures were used to estimate the percentage of questing adults on Fire Island that found deer hosts, excluding those that attached to other host species. Approximately 56% of these questing adult ticks found deer hosts in 1995 and 50% found deer hosts in 1996. Therefore, in areas where vertebrate hosts are highly abundant, large proportions of the questing tick population can find hosts. Moreover, comparisons of tick densities at different sites by flagging can be potentially biased by differences in host densities among sites.

Infection of Ixodes ricinus (Acari: Ixodidae) by Borrelia burgdorferi sensu lato in North Africa.

Free-living adult Ixodes ricinus L, were collected in Amdoun, situated in the Kroumiry mountains in northwestern Tunisia (North Africa). Using direct fluorescence antibody assay, the infection rate of field-collected I. ricinus by Borrelia burgdorferi sensu lato was 30.5% (n = 72). No difference in infection rate was observed between male and female ticks. Spirochetes that had been isolated from I. ricinus from Ain Drahim (Kroumiry Mountains) in 1988 were identified as Borrelia lusitaniae (formerly genospecies PotiB2). This is the first identification of a genospecies of Borrelia burgdorferi sensu lato from the continent of Africa.

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus).

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 x 10(5) cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase-PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

Reservoir competence of the meadow vole (Rodentia: Cricetidae) for the Lyme disease spirochete Borrelia burgdorferi.

The reservoir competence of the meadow vole, Microtus pennsylvanicus Ord, for the Lyme disease spirochete Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner was established on Patience Island, RI. Meadow voles were collected from 5 locations throughout Rhode Island. At 4 of the field sites, M. pennsylvanicus represented only 4.0% (n = 141) of the animals captured. However, on Patience Island, M. pennsylvanicus was the sole small mammal collected (n = 48). Of the larval Ixodes scapularis Say obtained from the meadow voles on Patience Island, 62% (n = 78) was infected with B. burgdorferi. Meadow voles from all 5 locations were successfully infected with B. burgdorferi in the laboratory and were capable of passing the infection to xenodiagnostic I. scapularis larvae for 9 wk. We concluded that M. pennsylvanicus was physiologically capable of maintaining B. burgdorferi infection. However, in locations where Peromyscus leucopus (Rafinesque) is abundant, the role of M. pennsylvanicus as a primary reservoir for B. burgdorferi was reduced.

Pathogenicity of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycetes) to Ixodes scapularis (Acari: Ixodidae).

The entomopathogenic fungus Metarhizium anisopliae is highly pathogenic to the black-legged tick, Ixodes scapularis. Spore concentrations of 10(8)/ml for engorged larvae and 10(7)/ml for engorged females resulted in 100% tick mortality, 2 wk postinfection. The LC50 value for engorged larvae (concentration to kill 50% of ticks) was 10(7) spores/ml. Metarhizium anisopliae shows considerable potential as a microbial control agent for the management of Ixodes scapularis.

Spontaneous occurrence of early region 1A reiteration mutants of type 5 adenovirus in persistently infected human T-lymphocytes.

Mutants of type 5 adenovirus (Ad5) with reiterated DNA sequences in the E1a region appeared in a human T-lymphocyte cell line, Molt-4, persistently infected with H5sub304, a deletion/substitution mutant that has a wild-type phenotype in viral replication. Endonuclease analyses and DNA sequencing revealed DNA reiteration in each mutant. In the four representative mutants investigated, the DNA reiterations all started within a six-base-pair consensus sequence, G(or C)CTGTG, located in the second exon of the E1a region (at nt 1333, 1367, or 1419). There was not any DNA homology between the breakpoints in the second exon and the inserting sequences (starting at nt 532, 710, or 792). Northern analyses suggested that the reiterated splicing sites of the representative mutants were all used in RNA splicing, and the closest donor and recipient joints were used most frequently. These observations imply that during persistent infection Ad5 underwent spontaneous mutations by sequence-specific breakage and nonhomologous end-end joining recombination events. These E1a reiteration mutants could be propagated in HeLa, A549, and KB cells; they were genetically stable; and they killed CREF cells at a strikingly high frequency. Preliminary observations tend to correlate this CREF cell killing with the accumulation of the early viral proteins and/or viral DNA in the infected cells. This degree of cell damage was not observed in Ad5wt or H5sub304 infection of CREF cells. The observed E1a reiterations provide a model to gain insight into understanding the evolutionary events of some, if not all, adenovirus types during many years of symbiotic, persistent relationship in human tonsils and adenoids and possibly other lymphoid organs.

Spatial distribution of larval Ixodes scapularis (Acari:Ixodidae) on Peromyscus leucopus and Microtus pennsylvanicus at two island sites.

Larval blacklegged ticks, Ixodes scapularis, were collected from white-footed mice. Peromyscus leucopus, on Prudence Island (where Microtus pennsylvanicus were not captured) and from meadow voles. M. pennsylvanicus, on Patience Island (where P. leucopus was absent) in Narragansett Bay, Rhode Island from June to October 1992. Ixodes scapularis larvae were also collected by flagging in the vicinity of host captures. On both islands, the relative density of larvae changed from July to September in samples from hosts, but not in flagging samples. Consequently, different sampling techniques can give different assessments of tick populations. Larvae were highly aggregated on both of the host species throughout the sampling period. As the mean relative density of larvae increased in the environment (based on flagging samples), larvae on the hosts became more dense and more crowded. Increased densities of larvae in the environment were not correlated with increased patchiness in the distribution of larvae among host animals on either island. Changes in the spatial distribution of larval I. scapularis on each host species had similar trends as larval densities and distributions within the environment. These results suggest that M. pennsylvanicus can serve as an alternative host for immature I. scapularis in a P. leucopus-free environment and have similar distributional characteristics.

The ups and downs of adenovirus vectors.

Owing to the detailed knowledge of the structure of the adenovirus virions, including their DNA genomes, especially types 2 and 5, they are convenient viruses for construction of vectors for gene therapy and vaccine immunization. It is critical to note, however, that adenoviruses produce pathogenic inflammatory responses to infection. The inflammation occurs even if the adenovirus does not replicate when the inoculum is sufficiently large, because only early gene expression is responsible for the pathogenic reaction. The inflammation consists of an early phase, in which tumor necrosis factor alpha (TNF-alpha) plays a major role, and a late phase consisting of an extensive T-cell response. It is important in the construction of adenovirus vectors not to delete a major portion of the early region 3 (E3) because: the E3 19 kD glycoprotein markedly reduces the capacity of the Class I major histocompatibility complex (Class I MHC) from transporting viral antigens to the surfaces of infected cells; and the E3 14.7 kD protein significantly inhibits the production of TNF-alpha and, therefore, reduces the polymorphonuclear response. Unfortunately the first generation of adenovirus gene therapy vectors contained large E3 deletions and, therefore, presented a significant safety problem. Subsequent adenovirus vectors consist of other deletions to overcome this difficulty.

Pathogenicity of Steinernema carpocapsae and S. glaseri (Nematoda: Steinernematidae) to Ixodes scapularis (Acari: Ixodidae).

The entomopathogenic nematodes Steinernema carpocapsae (Weiser) and S. glaseri (Steiner) are pathogenic to engorged adult, blacklegged ticks, Ixodes scapularis (Say), but not to unfed females, engorged nymphs, or engorged larvae. Nematodes apparently enter the tick through the genital pore, thus precluding infection of immature ticks. The timing of tick mortality, and overall mortality after 17 d, did not differ between infections by S. carpocapsae and S. glaseri. These nematodes typically do not complete their life cycles or produce infective juveniles in I. scapularis. However, both species successfully produced infective juveniles when the tick body was slit before nematode infection. Mortality of engorged I. scapularis females infected by S. carpocapsae was greater than uninfected controls, but did not vary significantly with nematode concentration (50-3,000 infective juveniles per 5-cm-diameter petri dish). The LC50 was 347.8 infective juveniles per petri dish (5 ticks per dish). Hatched egg masses of infected ticks weighed less than those of uninfected controls. Mortality of infected ticks was greatest between 20 and 30 degrees C, and was lower at 15 degrees C.