Projects

Ecology of Lyme disease in Texas.

In recent years increasing numbers of Lyme disease cases have been reported in Texas, a state considered Lyme disease free. These patients have been diagnosed with the disease after numerous tests, missing the time window in which antibiotic treatment is an effective measure to treat the disease. Therefore, most of the patients have developed chronic Lyme disease. It is the interest of our laboratory understanding the distribution of Lyme disease in the South of US, in areas that have been considered free for this disease. We are sampling ticks in areas classified as wet and dry in the state of Texas and analyzing the presence of Borrelia burgdorferi senso stricto (the causative agent of Lyme disease) by PCR (and consequent sequence). Together with Texas Parks and Wildlife management areas we are collecting ticks from deer, feral swine, jabalinas and any other exotic animal the hunters will bring to the management areas during the appropriate hunting seasons. By using this already set up infrastructure we will be able to determine the possibility of having other competent reservoirs and tick vectors for this bacterium in southern US.

We have already started the tick collection with help from different WMA, the Brazos Valley Animal Shelter, the CVM parasitology lab, volunteers from the Lyme disease support group in Houston and Austin and local veterinarians. In this process we have collected around 200 ticks from which about 34% were Ixodes scapularis (the deer tick, vector for the transmission of Lyme disease), 27% were Ammblyoma americanum (lonestar tick), 22% Dermacentor spp. and 17% Rhipicephalus sanguineus.

This is a work in progress. Nevertheless we have found that most of the Ixodes ticks received gave positive in 2 PCR reactions with 2 different targets (fla and the intergenic region 16SrRNA-23SrRNA (IGR)), and they were confirmed by sequencing of the IGR.
This results show evidence that B. burgdorferi, the causative agent of Lyme disease is present in Texas. Further studies need to be done in order to determine the animal species involved in maintaining the enzootic cycle of this pathogen and which arthropod vectors are involved in transmitting the disease to humans and other mammalian hosts. This is particularly interesting in areas where the Ixodes ticks has not been found (West Texas) but we still see veterinary and human Lyme disease cases.

Design of a pan-specific serological test for Lyme

The research proposed here addresses aspects of the roadmap proposed by the CDC to prevent infectious diseases like LD by sustaining the essentials and innovating the future, otherwise known as the CDC's ID Framework [4]. Currently, there are no epidemiological data available nationwide regarding the number of LD cases in domestic animals due to the fact that this is not a reportable disease in veterinary medicine in most states. The Texas Veterinary Medical Diagnostic Laboratory (TVMDL) tested a significant number of animals for LD. A total of 69 positive animal cases were identified, mainly horses and dogs, and occurred mostly in Eastern Texas. The number of confirmed human LD cases in Texas has been increasing considerably over the last 10 years (data provided by the Texas Department of State Health Services), but epidemiological information on the drivers behind that trend is missing. This situation impairs our ability to develop effective public health and veterinary preventive interventions. Most of the LD cases in Texas occurred in major metropolitan areas and primarily in Eastern Texas as observed in the animal cases. Human cases have also been reported in rural areas in West Texas and the Panhandle, where few to no animal cases have been reported. The differences in reporting between rural and metropolitan areas could be explained by a lack of information and awareness in the former. There are a number of commercially available serological diagnostic kits for LD to be used in animal and human health [5-9] , but they are relatively costly and mostly detect chronic cases with clinical signs. Additionally, these kits are species-specific and are impractical for use in surveillance programs for LD in species other than humans and canines, such as wildlife including exotic species. Economically accessible serodiagnostic kits that detect acute and subclinical cases are needed [5, 6, 8, 10-15] . The availability of reliable tests to detect B. burgdorferi represents another gap in the diagnostic armamentarium against LD [9].
The CDC's ID Framework promotes "One Health" approaches to prevent emergence and spread of zoonotic diseases like LD. Relative to our research program, the development of better diagnostic tests to be used in veterinary medicine for LD detection has a high impact on animal and human health. Better diagnostic tests will enable early intervention and increase the efficacy of treatments in animals and humans. A pan-specific diagnostic test will be of great value when establishing surveillance programs because it would avoid the generation of reagents for each specific animal species to be monitored. Comparisons of human and animal endemic areas will provide evidence of which animal species can serve as sentinels and identify high-risk areas of human LD, which could be targeted in public health awareness programs. This approach is expected to facilitate the creation of predictive models for areas where tick vectorsare likely to emerge, which ultimately would prevent the spread of LD.

Sequence B. burgdorferi transcriptome in the tick and mammalian host during transmission of Lyme disease.

The infectivity of B. burgdorferi depends on its ability to rapidly alter gene expression in response to highly disparate environmental signals following transmission from infected ticks. The only approach scientist have utilized to study this process is based on microarray analysis using in vitro conditions that mimic the pH and temperature in the tick during the transmission from the tick to the mammalian host or by using dialysis membranes inserted in the peritoneum of rats [16-20] . Others have analyzed the gene expression of a selected number of genes of B. burgdorferi in the tick vector by using real time PCR analysis. Next generation sequencing (NGS) technology offers the opportunity to profile the transcriptome of B. burgdorferi in the natural vector or the mammalian host. This approach overcomes limitations like the use of dialysis membranes to try to determine bacterial gene expression in the skin and other mammalian and tick tissues during the first hours post infection.
The CDC's ID Framework describes priority activities to achieve strong public health fundamentals, high‐impact interventions, and sound health policies [4]. Our program outlines research on surveillance, laboratory detection, and epidemiologic investigation to generate science-based information that can be used to prevent, detect, and control LD in human and animal populations. Specifically, the transcriptome of B. burgdorferi will be studied in the tick Ixodes scapularis during the first hours of pathogen transmission as a means to create knowledge for the development of a pan-specific LD diagnostic tool that could be used in veterinary medicine and surveillance programs. In contrast with previous studies [21, 22] , the use of NGS will facilitate the study of gene expression in B. burgdorferi before, during and after the tick blood meal. This study will provide significant information regarding the genes that orchestrate spirochete transmission from infected ticks to mammalian hosts during the early stages of infection. Moreover, this dynamic approach will provide potential targets for the development of therapeutics and vaccine candidates that will improve the diagnostics, surveillance and prevention of Lyme disease.
In addition, by developing this technique we will be able to find different Borrelial genospecies. This capability will be of great value for the study of the geographical distribution of Lyme disease in Texas and other areas of the country, and to characterize the type of infection that will cause each one of the isolates (arthritis versus carditis, etc). Furthermore, this project will develop a series of techniques that could be applicable not only to the study of Lyme disease but to other vector borne disease of importance in agriculture and/or public health.

VWFA containing proteins.

The ability of this spirochetal pathogen to colonize mammals is dependent on its ability to rapidly alter gene expression in response to highly disparate environmental signals following transmission from infected ticks. Some of the open reading frames (ORFs), up-regulated upon infection, are members of a large family of proteins termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), which facilitate adherence of B. burgdorferi to extra-cellular matrix components of the host. By comparative genome analysis, a family of von Willebrand factor A (VWFA) domain-containing proteins have been identified in B. burgdorferi [23-25] . These chromosomally encoded proteins (BB0172, BB0173, BB0175 and BB0325) could be involved in the interactions of B. burgdorferi with the mammalian host [25]. The VWFA domains present in extracellular matrix (ECM) proteins and on eukaryotic cells are involved in cell adhesion and protein-protein interactions (61, 90). Therefore, these borrelial proteins with VWFA domains might be involved in the adhesion of B. burgdorferi to eukaryotic cells, ECM components or to activated platelets and thus play a role in the virulence mechanisms of B. burgdorferi.
The objective of this project is to characterize biochemically and genetically the role of VWFA domain-containing proteins during borrelial infection in the murine model of Lyme disease. The central hypothesis is thatVWFA domain-containing proteins are critical for the interaction of B. burgdorferi with its host cells and matrices. This study will provide information on the borrelial determinants that facilitate interaction with ECM components and/or to integrins thereby facilitating colonization and dissemination during infection of the mammalian host.

Distribution of Lyme Disease in Texas. Cumulative cases from 2000 to 2010.

Life Microscopy of B burgdorferi

3D Image of B burgdorferi

Too Much Oxygen Kills Borrelia