Scott A. Bernhardt

Use of Real-Time PCR To Resolve Slide Agglutination Discrepancies in Serogroup Identification of Neisseria meningitidis

ABSTRACT Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia in children and young adults in the United States. Rapid and reliable identification of N. meningitidis serogroups is crucial for judicious and expedient response to cases of meningococcal disease, including decisions about vaccination campaigns. From 1997 to 2002, 1,298 N. meningitidis isolates, collected in the United States through the Active Bacterial Core surveillance (ABCs), were tested by slide agglutination serogrouping (SASG) at both the ABCs sites and the Centers for Disease Control and Prevention (CDC). For over 95% of isolates, SASG results were concordant, while discrepant results were reported for 58 isolates. To resolve these discrepancies, we repeated the SASG in a blinded fashion and employed ctrA and six serogroup-specific PCR assays (SGS-PCR) to determine the genetic capsule type. Seventy-eight percent of discrepancies were resolved, since results of the SGS-PCR and SASG blinded study agreed with each other and confirmed the SASG result at either state health laboratories or CDC. This study demonstrated the ability of SGS-PCR to efficiently resolve SASG discrepancies and identified the main cause of the discrepancies as overreporting of these isolates as nongroupable. It also reemphasized the importance of adherence to quality assurance procedures when performing SASG and prompted prospective monitoring for SASG discrepancies involving isolates collected through ABCs in the United States.

Burkholderia cepacia Complex in Cystic Fibrosis: Frequency of Strain Replacement during Chronic Infection

Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

Rapid Intraspecific Evolution of miRNA and siRNA Genes in the Mosquito Aedes aegypti

RNA silencing, or RNA interference (RNAi) in metazoans mediates development, reduces viral infection and limits transposon mobility. RNA silencing involves 21–30 nucleotide RNAs classified into microRNA (miRNA), exogenous and endogenous small interfering RNAs (siRNA), and Piwi-interacting RNA (piRNA). Knock-out, silencing and mutagenesis of genes in the exogenous siRNA (exo-siRNA) regulatory network demonstrate the importance of this RNAi pathway in antiviral immunity in Drosophila and mosquitoes. In Drosophila, genes encoding components for processing exo-siRNAs are among the fastest evolving 3% of all genes, suggesting that infection with pathogenic RNA viruses may drive diversifying selection in their host. In contrast, paralogous miRNA pathway genes do not evolve more rapidly than the genome average. Silencing of exo-siRNA pathway genes in mosquitoes orally infected with arboviruses leads to increased viral replication, but little is known about the comparative patterns of molecular evolution among the exo-siRNA and miRNA pathways genes in mosquitoes. We generated nearly complete sequences of all exons of major miRNA and siRNA pathway genes dicer-1 and dicer-2, argonaute-1 and argonaute-2, and r3d1 and r2d2 in 104 Aedes aegypti mosquitoes collected from six distinct geographic populations and analyzed their genetic diversity. The ratio of replacement to silent amino acid substitutions was 1.4 fold higher in dicer-2 than in dicer-1, 27.4 fold higher in argonaute-2 than in argonaute-1 and similar in r2d2 and r3d1. Positive selection was supported in 32% of non-synonymous sites in dicer-1, in 47% of sites in dicer-2, in 30% of sites in argonaute-1, in all sites in argonaute-2, in 22% of sites in r3d1 and in 55% of sites in r2d2. Unlike Drosophila, in Ae. aegypti, both exo-siRNA and miRNA pathway genes appear to be undergoing rapid, positive, diversifying selection. Furthermore, refractoriness of mosquitoes to infection with dengue virus was significantly positively correlated for nucleotide diversity indices in dicer-2.

Abundant nuclear copies of mitochondrial origin (NUMTs) in the Aedes aegypti genome

A portion of the Aedes aegypti mitochondrial NADH dehydrogenase subunit 4 gene (ND4) was amplified using PCR with a 42 °C annealing temperature. Amplified fragments from individual mosquitoes were similar to ND4 but contained multiple segregating sites. We suspected that nuclear copies of mitochondrial origin (NUMTs) exist in the Ae. aegypti genome. A BlastN search in VectorBase with the entire Ae. aegypti mitochondrial genome identified 233 NUMTs comprising 110 178 bp in 145 supercontigs. At a density of 0.080 bp/kb, this represents the second highest density of NUMTs in an insect genome and the highest in Diptera. Analyses of flanking sequences suggested that Ae. aegypti NUMTs arise through mtDNA leakage from damaged mitochondria followed by breakage and nonhomologous recombination, rather than through duplicative processes such as transposition or molecular drive.

Yersinia pestis infection and laboratory conditions alter flea-associated bacterial communities

We collected Oropsylla montana from rock squirrels, Spermophilus varigatus, and infected a subset of collected fleas with Yersinia pestis, the etiological agent of plague. We used bar-tagged DNA pyrosequencing to characterize bacterial communities of wild, uninfected controls and infected fleas. Bacterial communities within Y. pestis-infected fleas were substantially more similar to one another than communities within wild or control fleas, suggesting that infection alters the bacterial community in a directed manner such that specific bacterial lineages are severely reduced in abundance or entirely eliminated from the community. Laboratory conditions also significantly altered flea-associated bacterial communities relative to wild communities, but much less so than Y. pestis infection. The abundance of Firmicutes decreased considerably in infected fleas, and Bacteroidetes were almost completely eliminated from both the control and infected fleas. Bartonella and Wolbachia were unaffected or responded positively to Y. pestis infection.

Interactions among Symbionts of Oropsylla spp. (Siphonoptera: Ceratophyllidae)

ABSTRACT We used high-throughput DNA sequencing to explore bacterial communities of three species of Oropyslla fleas [Oropsylla hirsuta (Baker), Oropsylla montana (Baker), and Oropsylla tuberculata cynomuris (Jellison) ] and detected seven bacterial lineages related to known insect symbionts. No significant co-occurrence patterns were detected among bacterial lineages, but relative abundance data suggest that the two most common lineages (Bartonella and Rickettsiales) interact negatively. Furthermore, presence of these two lineages significantly reduced bacterial diversity within fleas.

Evidence of multiple chromosomal inversions in Aedes aegypti formosus from Senegal.

Chromosomal inversions are prevalent in mosquito species but polytene chromosomes are difficult to prepare and visualize in members of the tribe Aedinii and thus there exists only indirect evidence of inversions. We constructed an F1 intercross family using a P1 female from a laboratory strain of Aedes aegypti aegypti (Aaa) and a P1 male Aedes aegypti formosus (Aaf) from a strain collected from south‐eastern Senegal. Recombination rates in the F2 offspring were severely reduced and genotype ratios suggested a deleterious recessive allele on chromosome 3. The F2 linkage map was incongruent in most respects with the established map for Aaa. Furthermore, no increased recombination was detected in F5 offspring. Recombination rates and gene order were consistent with the presence in Aaf of at least four large inversions on chromosome 1, a single small inversion on chromosome 2 and three inversions on chromosome 3.

Assessing Insecticide Susceptibility of Laboratory Lutzomyia longipalpis and Phlebotomus papatasi Sand Flies (Diptera: Psychodidae: Phlebotominae)

ABSTRACT Chemical insecticides are effective for controlling Lutzomyia and Phlebotomus sand fly (Diptera: Psychodidae) vectors of Leishmania parasites. However, repeated use of certain insecticides has led to tolerance and resistance. The objective of this study was to determine lethal concentrations (LCs) and lethal exposure times (LTs) to assess levels of susceptibility of laboratory Lutzomyia longipalpis (Lutz and Nieva) and Phlebotomus papatasi (Scopoli) to 10 insecticides using a modified version of theWorld Health Organization (WHO) exposure kit assay and Centers for Disease Control and Prevention (CDC) bottle bioassay. Sand flies were exposed to insecticides coated on the interior of 0.5-gallon and 1,000-ml glass bottles. Following exposure, the flies were allowed to recover for 24 h, after which mortality was recorded. From dose-response survival curves for L. longipalpis and P. papatasi generated with the QCal software, LCs causing 50, 90, and 95% mortality were determined for each insecticide. The LCs and LTs from this study will be useful as baseline reference points for future studies using the CDC bottle bioassays to assess insecticide susceptibility of sand fly populations in the field. There is a need for a larger repository of sand fly insecticide susceptibility data from the CDC bottle bioassays, including a range of LCs and LTs for more sand fly species with more insecticides. Such a repository would be a valuable tool for vector management.

Distribution and Habitat of Ixodes pacificus (Acari: Ixodidae) and Prevalence of Borrelia burgdorferi in Utah

ABSTRACT Knowledge about the distribution and abundance of the western black-legged tick, Ixodes pacificus Cooley and Kohls, in Utah is limited. Recent concerns over tick-borne diseases in Utah, primarily Lyme disease, have reinvigorated the need to understand the distribution and habitats favored by this tick species. We surveyed 157 sites throughout Utah to examine the distribution, abundance, and habitat of I. pacificus. In total, 343 adult ticks were collected from 2011 to 2013. Specifically, 119 I. pacificus, 217 Dermacentor andersoni Stiles, six D. albipictus Packard, and one D. hunteri Bishopp were collected. Overall, tick abundance was relatively low in the areas evaluated in Utah. I. pacificus collections were limited to sites above 1700 m. Ninety-two percent of I. pacificus were captured in the Sheeprock Mountains in Tooele County. I. pacificus positive collection sites were characterized by Gambel oak (Quercus gambelii Nuttall), juniper (Juniperus spp. L.), big sagebrush (Artemisia tridentata Nuttall) and black sagebrush (A. nova Nelson), and mixed grass habitat. All I. pacificus ticks were tested for the presence of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner, sensu stricto) using real-time PCR. All ticks tested negative for B. burgdorferi. The likelihood of encountering I. pacificus and acquiring Lyme disease in the areas evaluated in Utah is considerably low due to low tick abundance and limited distribution, as well as low prevalence (or absence) of B. burgdorferi in Utah.

Novel Strategies to Control Aedes aegypti and Dengue

Vector-borne diseases are resurgent throughout the world. There are critical needs to develop novel approaches and strategies and improved public health capacity to prevent and control these diseases. Dengue is an archetypical resurging and emerging disease. At the Arthropod-Borne and Infectious Diseases Laboratory, we are investigating novel strategies for Aedes aegypti and dengue control; including development of (1) transgenic Ae. aegypti that are innately immunologically resistant to dengue virus infection, (2) a Casa Segura (safe house) approach based upon long-lasting insecticide-treated materials to protect humans from Ae. aegypti in the epidemiologically most important point of contact – the home, and (3) a dengue decision support system to enhance the efficacy of Ae. aegypti and dengue control programs. The results provide promise for targeting the vector to prevent and control dengue.