A METAGENOMIC STUDY OF THE TICK MIDGUT

A Metagenomic Study of the Tick Midgut Daniel Yuan, B.S. Supervisory Professor : Steven J. Norris, Ph.D. Southern tick–associated rash illness (STARI) or Master’s disease is a Lyme-like illness that occurs following bites by Amblyomma americanum, the lone-star tick. Clinical symptoms include a bull’s eye rash similar to the erythema migrans lesions of Lyme disease, as well as fever and joint pains. Lyme disease is caused by Borrelia burgdorferi and related spirochetes. However, B. burgdorferi has not been detected in STARI patients, or in ticks in the South Central U.S. The causative agent of STARI has not been identified, although it was once thought to be caused by another Borrelia species, Borrelia lonestari. Furthermore, while adult A. americanum have up to a 5.6% Borrelia lonestari infection rate, the prevalence of all Borrelia species in Texas ticks as a whole is not known. Previous studies indicate that 6%-30% of Northern Ixodes scapularis ticks are infected by Borrelia burgdorferi while only 10% of Northern A. americanum and I. scapularis ticks are infected by Borrelia species. The first specific aim of this project was to determine the bacterial community that inhabits the midgut of Texas and Northeastern ticks by using high throughput metagenomic sequencing to sequence bacterial 16S rDNA. Through the use of massively parallel 454 sequencing, we were able to individually sequence hundreds of thousands of 16S rDNA regions of the bacterial flora from 133 ticks from the New York, Missouri and Texas. The presence of previously confirmed endosymbionts, specifically the Rickettsia spp. and Coxiella spp., that are commonly found in ticks were confirmed, as well as some highly prevalent genera that were previously undocumented. Furthermore, multiple pathogenic genera sequences were often found in the same tick, suggesting the possibility of co-infection of multiple pathogenic species. The second specific aim was to use Borrelia specific primers to screen 344 individual ticks from Missouri, Texas and the Northeast to determine the prevalence of Borrelia species in ticks. To screen for Borrelia species, two housekeeping genes, uvrA and recG, were selected as well as the 16S-23S rDNA intergenic spacer. Ticks from Missouri, Texas and New York were screened. None of the Missouri or Texas ticks tested positive for Borrelia spp. The rate of I. scapularis infection by B.burgdorferi is dependent on tick feeding activity as well as reservoir availability. B. burgdorferi is endemic in the Northeast, sometimes reported as highly present in over 50% of all I. scapularis ticks. 11.6% of all New York ticks were positive for a species of Borrelia, however only 6.9% of all New York ticks were positive for B. burgdorferi. Despite being significantly lower than 50%, the results still fall in line with previous reports of about the prevalence of B. burgdorferi. 1.5% of all Texas ticks were positive for a Borrelia species, specifically B. lonestari. While this study was unable to identify the causative agent for STARI, 454 sequencing was able to provide a tremendous insight into the bacterial flora and possible pathogenic species of both the I. scapularis and the A. americanum tick.

The dynamic proteome of Lyme disease Borrelia.

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

The seroprevalence of Rocky Mountain spotted fever in shelter dogs in Harris County

Rocky Mountain spotted fever (RMSF) is a tick-borne illness caused by the bacteria Rickettsia rickettsii, with infections occurring in humans and dogs. The prominent tick vector of RMSF, Dermacantor variabilis, and another potential vector, Rhipacephalus sanguineus, are prevalent in Texas. The goal of this study was to determine the prevalence of past infections by testing for IgG antibodies to R. rickettsii in dogs in an animal shelter in Harris County using an immunofluorescence assay (IFA) test. We found that 12.6% (24) of 191 dogs tested had a positive IFA test at 1:64 serum dilution, indicating infection at some time in the past. We also sampled the ticks present on dogs in the animal shelter to understand the prevalence of potential vector species. Of a total of 58 ticks, 86% were D. variabilis and the remaining 14% were R. sanguineus. The results of this study demonstrate that RMSF has the potential to be, and may already be, endemic to the Harris County area. Public health actions such as heightened surveillance and education that RMSF is present would be appropriate in the Harris County area.^

Plasmid -encoded virulence determinants of Borrelia burgdorferi

Borrelia burgdorferi, a spirochete and the causative agent of Lyme disease, infects both mammals and ticks. Its genome, sequenced in 1997, consists of one linear chromosome and over 20 linear and circular plasmids. Continuous passage of organisms in culture causes them to lose certain plasmids and also results in loss of infectivity in mammals. In this work, 19 B. burgdorferi clonal isolates were examined for infectivity in mice and for plasmid content utilizing polymerase chain reaction (PCR). Two plasmids, a 28 kilobase (kb) linear plasmid (Ip28-1) and a 25 kb linear plasmid (Ip25) were found to be required for full infectivity. Previous studies had demonstrated that Ip28-1 contains the vls locus, which is involved in antigenic variation and immune evasion. Gene BBE22 on Ip25 is predicted to encode the nicotinamidase PncA, an enzyme that converts nicotinamide to nicotinic acid as part of a pathway for NAD synthesis. To examine the potential role of BBE22 in infectivity, a shuttle vector containing BBE22 (pBBE22) was constructed and used to transform B. burgdorferi clone 5A13, which contains all plasmids except lp25. Transformation with pBBE22 restored infectivity of clone 5A13 in mice, whereas 5A13 transformed with the shuttle vector alone was not infectious. To determine whether BBE22 acts as a nicotinamidase in vivo, a Salmonella typhimurium pncA− nadB− transposon mutant was transformed with pBBE22 or with pQE30:BBE22, which contained BBE22 in an E. coli expression vector. Both constructs complemented the Salmonella mutant, permitting growth in minimal media plus nicotinamide. Salmonella cells over-expressing BBE22 also exhibited nicotinamidase activity, as determined by ammonia production in the presence of nicotinamide. Site-directed mutagenesis of BBE22 at the predicted active site (resulting in a Cys120Ala substitution) abrogated the ability to restore infectivity to B. burgdorferi 5A13 and to complement the pncA mutation in S. typhimurium. These studies indicate that BBE22 is a nicotinamidase required for NAD synthesis and survival of B. burgdorferi in mammals. This is also the first demonstration of ‘molecular Koch's postulates’ in B. burgdorferi, i.e. that a specific gene is essential for infectivity of the Lyme disease spirochete. ^