Rickettsia and endosymbiont interference and epidemiology within Longitarsus flea beetle and Dermacentor occidentalis tick hosts.

Rickettsiae are small, gram negative, rod-shaped, obligate intracellular, endosymbiotic alphaproteobacteria that are responsible for several of the oldest zoonoses known to man. Over the past 10 years, genetic analysis of Rickettsiae found in arthropod vectors, mammal reservoirs and clinical disease specimens has resulted in the recognition of new pathogenic rickettsial species and a re-evaluation of disease caused by species previously thought to be non-pathogenic, including, most recently Rickettsia philipii strain 364D. Interference between different Rickettsia species co-infecting ticks has been described, although, the mechanisms are unknown. Intereference is thought to lead to dramatic epidemiological consequences such as defining the geographic distribution of disease. In this dissertation, the association and possible interference between Rickettsia spp. and other bacteria was investigated using two different invertebrate models, namely, insect flea beetles of the Longitarsus genus and the arachnid tick Dermacentor occidentalis. Longitarsus flea beetles are herbivorous and complete their entire life cycle on and around an individual plant and its soil, in contrast to Dermacentor ticks that imbibe blood from three different vertebrate hosts to complete their life cycle. PCR amplification and Sanger sequencing of rickettsial gene sections and PCR of 16S rRNA gene segments followed by next generation sequencing, respectively, were used to identify rickettsias and other bacteria that constituted the microbiome of these invertebrate hosts. The Quantitative Insights Into Microbial Ecology (QIIME) open source bioinformatics pipeline was used for sequence data analysis and to explore the different intermicrobial and microbe-host relationships of Rickettsia. We found that while the species of Longitarsus directed the make-up of their Rickettsia and Wolbachia endosymbionts, in contrast, in Dermacentor ticks, other Francisella-like endosymbionts and, to a lesser extent, non-endosymbiotic organisms appeared to “interfere” with rickettsial infection. An additional goal of this investigation was to describe the prevalence and distribution of Rickettsias in Dermacentor occidentalis in San Diego County. Both pathogenic and nonpathogenic Rickettsias were detected in ticks collected from different locations over several years. These findings are described in the dissertation that follows

D. occidentalis 16S rRNA OTU filtered and rarefied OTU table

D. occidentalis 16S rRNA OTUs after filtering for samples with at least 150 OTUs, no single OTUs, OTUs occur in at least 10 samples and rarefied to 1500 even sampling depth

Rickettsia and endosymbiont interference and epidemiology within Longitarsus flea beetle and Dermacentor occidentalis tick hosts.

Rickettsiae are small, gram negative, rod-shaped, obligate intracellular, endosymbiotic alphaproteobacteria that are responsible for several of the oldest zoonoses known to man. Over the past 10 years, genetic analysis of Rickettsiae found in arthropod vectors, mammal reservoirs and clinical disease specimens has resulted in the recognition of new pathogenic rickettsial species and a re-evaluation of disease caused by species previously thought to be non-pathogenic, including, most recently Rickettsia philipii strain 364D. Interference between different Rickettsia species co-infecting ticks has been described, although, the mechanisms are unknown. Intereference is thought to lead to dramatic epidemiological consequences such as defining the geographic distribution of disease. In this dissertation, the association and possible interference between Rickettsia spp. and other bacteria was investigated using two different invertebrate models, namely, insect flea beetles of the Longitarsus genus and the arachnid tick Dermacentor occidentalis. Longitarsus flea beetles are herbivorous and complete their entire life cycle on and around an individual plant and its soil, in contrast to Dermacentor ticks that imbibe blood from three different vertebrate hosts to complete their life cycle. PCR amplification and Sanger sequencing of rickettsial gene sections and PCR of 16S rRNA gene segments followed by next generation sequencing, respectively, were used to identify rickettsias and other bacteria that constituted the microbiome of these invertebrate hosts. The Quantitative Insights Into Microbial Ecology (QIIME) open source bioinformatics pipeline was used for sequence data analysis and to explore the different intermicrobial and microbe-host relationships of Rickettsia. We found that while the species of Longitarsus directed the make-up of their Rickettsia and Wolbachia endosymbionts, in contrast, in Dermacentor ticks, other Francisella-like endosymbionts and, to a lesser extent, non-endosymbiotic organisms appeared to “interfere” with rickettsial infection. An additional goal of this investigation was to describe the prevalence and distribution of Rickettsias in Dermacentor occidentalis in San Diego County. Both pathogenic and nonpathogenic Rickettsias were detected in ticks collected from different locations over several years. These findings are described in the dissertation that follows

Percent abundance of bacteria taxa within Longitarsus samples.

Percent abundance of bacteria taxa found within Longitarsus flea beetle sample from 3 locations in Germany as identified by Illumina sequencing of the 16S rRNA gene V4 region

Dermacentor occidentalis 16S rRNA OTU table

OTU table (from 515F/806R 16S rRNA gene sequences) derived from individual Dermacentor occidentalis ticks via Illumina sequencing. OTU table was filtered to remove samples with less than 150 sequences and singleton OTUs and was rarefied to an even sampling depth of 150

Dermacentor occidentalis microbiome 16S rRNA gene sequences from San Diego County

Demultiplexed 16S rRNA gene sequences derived from individual Dermacentor occidentalis ticks from 4 different areas in San Diego County in 2014. Sequenced on an Illumina MiSeq platform

Longitarsus 16S rRNA microbiome sequences

<p>This is the gzipped 16S rRNA gene sequence file of 3 <em>Longitarsus</em> flea beetle species: <em>L. luridis, L. melanocephalus, </em>and<em> L. pratensis</em> generated by Illumina MiSeq pyrosequencing.</p

Mapping file for Longitarsus 16S rRNA microbiome sequences

<p>This is the mapping file for the Illumina MiSeq pyrosequenced 16S rRNA gene microbiome of 3 <em>Longitarsus</em> flea beetle species: <em>L. luridis, L. melanocephalus, </em>and<em> L. pratensis</em>.</p

Raccoons in San Diego County as Sentinels for West Nile Virus Surveillance

OBJECTIVE: To investigate the potential of utilizing raccoons as sentinels for West Nile Virus (WNV) in an effort to guide public health surveillance, prevention, and control efforts. INTRODUCTION: Since its detection in 1999 in New York, WNV spread westward across the continent, and was first detected in California in 2003 in Imperial County (1). In California and in many states, birds, especially corvids, are used as sentinel animals to detect WNV activity. Recent seroprevalence studies have shown WNV activity in different wild mammalian species (1–3); in the United States, WNV sero-prevalence in some studies in raccoons has ranged from 34–46% (3,4). In addition, it has been shown that after experimental infection, raccoons can attain high viral titers and shed WNV in their saliva and feces (5). Given their peridomestic nature, we investigated the feasibility of their use as sentinels for early warning of WNV and as indicators of WNV activity as a strategy to better localize WNV transmission foci in guiding vector control efforts. METHODS: Sick, injured or orphaned raccoons undergoing rehabilitation at Project Wildlife, one of the largest, non-profit wildlife rehabilitation organizations in the United States, located in San Diego County, were tested for WNV shedding. Project Wildlife team members who regularly care for sick, injured, or orphaned raccoons were trained to collect oral and fecal samples for viral testing during 2011 and 2012 upon raccoons’ arrival to Project Wildlife. Oral and fecal samples were tested using real-time PCR for the envelope gene of WNV. RESULTS: To date 71 raccoons have been tested for WNV and all PCR test results have been negative. Of the 71 raccoons tested from May 2011 to October 2011 and June 2012 to September 2012, 85.9% (n=61) had age classification data. The majority of these raccoons were young; 52.5% (n=32) were days or weeks old and 39.3% (n=24) were classified as juveniles. All raccoons were found primarily in urban settings at least 20 miles from the northern edge of the County. CONCLUSIONS: While none of the raccoon samples tested in this study were found to be WNV positive, surveillance data from San Diego County suggests that WNV activity during this time period was extremely low. From January–October 2011, San Diego County Vector Control reported all negative results for WNV in dead birds, sentinel chickens, horses, and humans for WNV; only 1 mosquito pool from the northern border region of the County tested positive for WNV (6). Thus, despite WNV activity throughout the state of California, the virus did not appear to be circulating widely in San Diego County in 2011 (7). To date during the 2012 season, San Diego County reported all negatives for WNV in dead birds, sentinel chickens, mosquito pools, and horses; only one human case of WNV was identified in an asymptomatic male during a routine blood donation (6). Further evaluation is needed to determine if raccoons are useful sentinel species for WNV surveillance. Testing should continue to evaluate if raccoons may serve as a more effective early warning sentinel for WNV than birds which can travel long distances from the exposure site, and to determine if raccoons may allow better localization of WNV activity