Lyme Grant Report Summary
Vector borne diseases are those infectious diseases that are transmitted to their animal host through the bite of an arthropod, most commonly an insect, or tick. Ticks can carry a variety of different zoonotic diseases (pathogens that are able to infect both human and other animal hosts), including those caused by bacteria. One zoonotic disease that is particularly prevalent in southeastern Massachusetts is Lyme disease, which is caused by the bacteria Borrelia burgdorferiThe prevalence of Lyme disease in the United States has continued to grow with confirmed human cases increasing from 10,000 in 1995 to over 27,000 in 2015 (CDC). More recently, other bacterial pathogens have been identified that are transmitted via tick bites, specifically the pathogen Anaplasma, which is the causative agent of Anaplasmosis and is frequently co-transmitted with B. burgdorferi.
Figure 1. Overview of the polymerase chain reaction. This technique is used to make billions of copies of a target gene
In our canine companions, routine testing for these pathogens involves assaying for antibodies against these organisms that are present in the blood. Previous research has shown that antibodies can persist for months or even years. As such, the original goal of this project was to assess how accurate these tests were at identifying active infections in dogs. To this end, dog blood samples collected at Marion Animal Hospital in Marion, MA were subjected to total community DNA extraction by means of enzymatic lysis, mechanical lysis, and chemical lysis. Extraction products were then subjected to PCR amplification (Figure 1) using two separate primer sets. One primer set targeted the bacterial 16S rRNA (small ribosomal subunit) gene (to assess for the presence of pathogens), while the other universal primer set targeted the small ribosomal subunit from both bacteria and eukaryotic (dog WBCs) organisms. Initially, these extractions proved to be unsuccessful. Several modifications were made to the protocol allowing for successful amplification of the eukaryotic small subunit rRNA gene primer set, however we were still unable to obtain PCR products using the bacterial specific primers.
Based upon these results, I developed three hypotheses:
  1. There were no bacterial pathogens present in the blood samples.
  2. The bacterial pathogens were present, but at such a low number our current assay was not sensitive enough to detect them.
  3. For Borrelia burgdorferi, the organism had already moved from the bloodstream into the tissues, and as such was not present in the samples.
Recently I revisited this part of the project making modifications to both the DNA extraction and the PCR amplification protocols. From the 12 blood samples subjected to these modified protocols, I was able to successfully amplify bacterial DNA (using 16S rRNA gene primers targeting bacterial DNA) from the blood samples that tested positive for one or both of the bacterial pathogens in question based on traditional antibody-antigen tests, while those dogs that had tested negative for these pathogens had no amplification products. This is a very limited data set (n=12), however this limited data suggests that the current testing methods are accurate in identifying active infections. With optimization of both the DNA extraction and the PCR amplification protocols I will be able to complete analysis of the remaining blood samples, and use these samples in PCR assays with primers specific for the pathogens in questionAdditionally, I will be renewing my IACUC protocol, which will allow Marion Animal Hospital to begin collecting additional blood samples for testing.
A second, and perhaps more important goal of this project is to better understand the epidemiology of B. burgdorferi and Anaplasma spp. in Ixodes scapularis ticks collected from Southeastern Massachusetts. Both of these agents are considered emerging tick-borne zoonotic agents of public health importance. Lyme disease and Anaplasmosis present as clinical syndromes ranging from asymptomatic to fatal diseases. More recently, it has been shown that patients treated for Lyme disease can suffer from Post-Treatment Lyme disease Syndrome or PTLDS. This chronic manifestation of the symptoms of Lyme disease is believed to be caused by tissue damage by the pathogen or damage to the body caused by the immune response to the infectious agent. Finally, individuals and dogs that have gone undiagnosed for either of these pathogens can develop severe, debilitating sequalae including cardiovascular disease and neurological impairment. Given the gravity of these diseases, it is critical to understand the prevalence of these pathogens in their tick vectors to gain a better understanding of the potential to contract these diseases if bitten by an Ixodes scapularis tick. There is currently a large repository of data on the incidence of these diseases and the prevalence in ticks for Barnstable and Dukes counties on Cape Cod, but there is little data for Plymouth County. This lack of data is shocking given the fact that several towns in Plymouth County have shown an incidence rate of infection of Lyme disease similar to that of towns in Barnstable and/or Dukes counties (personal communication).
When this project began there was a severe drought in Southeastern Massachusetts, which hindered our attempts to collect a significant number of ticks in Plymouth County. However, there was an abundance of ticks still present on the Cape. Given this, ticks were collected from various locations in Barnstable County. The GPS coordinates were recorded, and ticks were preserved in 100% ethanol. In the lab, the ticks were identified to genus and species, aged (adult/nymph/larvae) and sexed (if an adult). Total community DNA extraction protocols were optimized over the course of 4 months. The final protocol involved ticks being cut with a sterile scalpel into three to five pieces. The ticks were then homogenized using a mechanical pestle. DNA was extracted using the Qiagen DNeasy Tissue and Blood kit according to manufacturers protocol with the following modifications. Samples were incubated in Enzymatic Lysis Buffer with lysozyme at a final concentration of 20 mg/ml for 1.5 hours. Samples were then incubated with Proteinase K for 1.5 hours. DNA extraction samples were then quantified using the Nanodrop 2000. 

Total community DNA was extracted from 112 of the ticks collected from Barnstable County. This DNA was then subjected to PCR amplification using primers specific for the 16S rRNA gene (8F/1492R), which is found in all bacteria. Gel electrophoresis was used to visualize the amplification products. Initial PCR amplifications resulted in no amplification products visualized on the gel. In order to overcome this, Bovine Serum Albumin (BSA) was added to the PCR amplification reactions. BSA has been shown to nonspecifically bind potential inhibitors of DNA polymerase, thus allowing amplification to proceed. Once BSA was added to these reactions, amplification products were obtained. With our PCR protocol optimized, we then utilized these DNA extractions in PCR amplification assays with primers specific for the pathogen B. burgdorferi, the causative agent of Lyme disease. Preliminary data suggests that approximately 40% of these samples tested positive for B. burgdorferi. This data is congruent with previous data reported for the prevalence of B. burgdorferi in I. scapularis ticks in Barnstable County (personal communication). Additional PCR amplifications will be run in order to confirm this data.
More recently, ticks have been collected from multiple towns in Plymouth County including Rochester, Lakeville, Middleboro, Halifax and Bridgewater. To date, DNA extractions from 75 ticks collected in Rochester have been conducted. The GPS coordinates were recorded, and ticks were preserved in 100% ethanol. In the lab, the ticks were identified to genus and species, aged (adult/nymph/larvae) and sexed (if an adult). Once again, extracted total community DNA was utilized in PCR reactions using the bacterial specific primers 8F and 1492R to confirm successful bacterial DNA extraction. Once this was confirmed, these samples were utilized in PCR amplification assays with primers specific for both B. burgdorferi and Anaplasma spp. In order to assess specificity of the primer sets for the particular pathogen in question, DNA was extracted from a suite of both Gram-positive bacteria (Bacillus subtilis, Streptococcus epidermidis, Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Escherichia coli and Proteus vulgaris). Two different, previously published primer sets were utilized to assess for the presence of B. burgdorferi. Neither of these primer sets had any cross-reactivity with the above mentioned bacteria, demonstrating that they were specific for the pathogen in question. Of the 75 ticks assayed, ~43% of them tested positive via PCR for B. burgdorferi.

Unfortunately, previously published primer sets for Anaplasma spp. proved to be less specific. One set of primers resulted in slight cross-reactivity with E. coli. This primer set did give stronger bands in the PCR for ~10% of samples. This data aligns well with infectivity rates of Anaplasma in ticks seen on the Cape. The second primer set gave a 100% infectivity rate. In order to confirm the prevalence of ticks infected with Anaplasma spp., additional, more specific, primer sets will be developed and tested.
Thanks to this grant, we are beginning to understand the prevalence of these emerging pathogens in Plymouth County, and the results are dire indeed for both humans and their canine companions. Fortunately, Plymouth County will be hiring an entomologist, with whom I am hoping to collaborate to continue this research and disseminate this information to the public. The future work on this project has no limits. During the summer I will continue to collect ticks from towns in Plymouth County and identify those infected with B. Burgdorferi. New primers will be designed and tested to confirm the data obtained for Anaplasma spp. and all of this data will be confirmed via real-time PCR assays. In addition, we will begin to assess for the protozoal pathogen Babesia spp. which is also considered an emerging pathogen and co-transmitted with Borrelia and Anaplasma spp.. Finally, moving forward, all nucleic acid extractions will involve DNA and RNA extractions to allow for RT-PCR assays of the retrovirus, Powassan virus, which is also transmitted by Ixodes scapularis and a threat to both canines and humans. It is my intent to continue to submit updates to the Board of Directors of Paws To People, and to include Paws To People on any grants or publications that come from this research. 

Respectfully submitted,
Jenna Mendell, PhD