Publication/Presentation Date

7-25-2014

Abstract

Abstract

Ixodes scapularis are vectors for human pathogens including Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia microti, and the emerging infectious agent, Borrelia miyamotoi. The goal of this study was to determine the prevalence of these tick-borne pathogens in deer ticks in the Lehigh Valley of eastern Pennsylvania, where tick populations are abundant. By doing so, the epidemiological risks to humans could be assessed and this information used to guide health care providers in the region. To be sensitive and specific in testing for these pathogens, real-time PCR was performed on DNA extracted from ticks. Nested conventional PCR and sequencing were also used when increased specificity was necessary. In 428 collected ticks, B. burgdorferi had the highest infection rate of 23.6%, followed by that of A. phagocytophilum (1.9%). The prevalence of B. microti and B. miyamotoi was less than 1%. Only the deer variant (AP-1) of the A. phagocytophilum was found so far and another species that is nonpathogenic to humans, Babesia odocolei, was found in a different sample. Detecting human pathogens required application of different methods to obtain more accurate results that will impact residents in the Lehigh Valley.

Introduction

Increased public awareness of tick-borne infections in the United States has raised concerns about the risk they pose, especially to those living in regions, such as the northeast, where tick populations are high. Commonly known as blacklegged or deer ticks, Ixodes scapularis are known vectors of the pathogens that cause human infections including Human Granulocytic Anaplasmosis and, the most common, Lyme Disease. The symptoms of these infections can be nonspecific and therefore difficult to diagnose. Assessing the prevalence of human pathogens in deer ticks can guide health care providers in time-sensitive clinical decision-making regarding prophylaxis and treatments. The Lehigh Valley of eastern Pennsylvania, an important source of medical care in the region, has an abundant deer tick population and thus is an area worth investigating (Duik-Wasser et al., 2012).

Lyme Disease is caused by the bacterial spirochete Borrelia burgdorferi (sensu lato). Those infected may experience cardiac, neurological, musculoskeletal, and dermatological complications, including the appearance of the characteristic bulls-eye rash (Bacon, Kiersten, & Mead, 2008). Lyme Disease is endemic in states of the upper Midwest and northeast, where 95% of the nation’s confirmed cases were reported in 2011 (Adams et al., 2013). Increased residence in suburban areas and reservoir host population expansion help explain the observed rise in reported cases of Lyme Disease in the nation, which was consistent from 1992-2006 (Bacon et al., 2008). Pennsylvania’s reported cases of Lyme Disease constituted 19% of the nation’s total in 2011. While the geographic distribution of cases is varied among Pennsylvania’s counties, many are reported in the eastern part of the state (Courtney et al., 2003). Incidence of Lyme disease in Lehigh County is 47 cases per 100,000, a rate above the average of Pennsylvania of 39.1 cases per 100,000 (Pennsylvania Department of Health, 2014). Studies have screened deer ticks in nearby counties for the prevalence of human pathogens, but none have been done in Lehigh County.

The geographic distribution of cases of Human Granulocytic Anaplasmosis (HGA) is similar to that of Lyme Disease. A strain of the intracellular bacterium Anaplasma phagocytophilum (ha variant) causes infection in humans by targeting the neutrophils. This results in fever, nausea, and related symptoms, but rarely causes death (Dumler et al., 2005). Incidence of HGA per million persons has risen from 1.4 cases in 2000 to 6.1 cases in 2010, indicating its emergence in the United States (Centers for Disease Control and Prevention [CDC], 2013a). In 2011, six cases of HGA were reported in Pennsylvania (CDC MMWR 2013-2).

Most human cases of babesiosis in the United States are caused by Babesia microti, a protozoan parasite that infects the red blood cells. While healthy individuals display no symptoms when infected, immunocompromised, elderly, and asplenic individuals can experience a malaria-like infection and serious complications including splenomegaly and hemolytic anemia (Teal, Habura, Ennis, Keithly, & Madison-Antenucci, 2012). Approximately 96% of the reported cases in 2011 came from 7 states, all of which were in the northeast and upper Midwest. While Pennsylvania does not require reporting of cases of babesiosis, 21 cases of babesiosis were voluntarily reported from 2005 to 2010, 90% of which came from residents in eastern Pennsylvania (Pennsylvania Department of Health, 2011). Babesiosis is emerging in the Lehigh Valley area where three cases of babesiosis were reported in 2013.

Borrelia miyamotoi causes tick-borne relapsing fever and has been detected recently in deer ticks in North America. Though currently rare, the infection is transmitted vertically in deer ticks meaning it can be spread quickly and thus, it is important to study the prevalence of this emerging infection in deer ticks.

This study involved the screening of deer ticks in the Lehigh Valley for human pathogens, specifically B. burgdorferi, A. phagocytophilum, B. microti, and B. miyamotoi, to determine the risk of infection that residents in the Lehigh Valley face when bitten in this region. Real-time Polymerase Chain Reaction (PCR) was used to detect the pathogens because it allows for specificity and sensitivity in screening, avoiding false negative and positive results.

Conventional PCR and sequencing were used in addition to real time PCR for discrimination between closely related species to increase specificity in the reported results.

Methods

Tick Collection: More than 428 ticks were collected from sites across Lehigh County. Sites of collection were accessible to the public and were suburban, grassy, and wooded areas. Ticks were collected using the dragging method. White dragging cloths were checked every few minutes for the presence of ticks and when found, they were removed and stored in vials containing 70% ethanol. Collected ticks were transported back to Muhlenberg College in Allentown, PA and identified by species, sex, and developmental stage (nymph/adult) before being stored in a -20°C freezer until use.

DNA Isolation: DNA extraction, real time PCR, and conventional PCR with the use of gel electrophoresis were conducted in separate rooms with the use of aerosol resistant tips and designated pipettes. A no-template control was prepared with each extraction as a precaution against contamination with tick or pathogen DNA. Before DNA extraction, each tick was placed in an individual bead tube containing yttria/zirconia beads (750 mg of 2.0 mm beads and 150 mg of 0.2 mm of beads). Adult ticks were sometimes bisected and one half was stored at -80°C in 70% ethanol, while the other was used in the extraction. DNA was extracted using the MoBIO DNA extraction kit according to the manufacturer’s protocol or the Qiagen QIAmp DNA Mini Kit with modifications to the procedure used in Crowder et al. (2010). Bead beading was used twice for vigorous shaking of the samples in a BioSpec Mini Bead Beater 16 (BioSpec, Bartlesville, OK) at top speed.

To determine the quality of the DNA from the extractions, 260/280 readings were taken of adult tick samples using the Nanodrop 2000c (Thermo Scientific). To affirm the presence of tick DNA in all samples, especially in nymphs that are below the sensitivity of the Nanodrop 2000c, an ITS-2 probe specific for Ixodes ticks was adapted from Strube, Montenegro, Epe, Eckelt, and Schnieder 2010. Since the ticks were visually identified as Ixodes Scapularis prior to the extractions, there was no need to distinguish between Ixodes species. The conventional PCR method of Norris, Klompen, Keirans, and Black (1996) was used to determine the presence of DNA in some of the samples. A possible correlation between real time PCR results and the concentration of DNA in tick samples after extraction is currently being investigated.

PCR Analysis: Taqman® assays containing AmpErase® were used to avoid contamination with amplified PCR product. Real time PCR reactions were set up in a laminar flow PCR hood (Labconco). Using the fast cycling program and Taqman® Advanced Master Mix with AmpErase®, 20 microliter reactions each containing 2 microliters of template DNA were carried out in an Applied Biosystems StepOne Plus thermal cycler. A two minute incubation period at 50°C initiated the cycling program to activate Uracil N-glycosylase, followed by 20 seconds at 95°C, and then 60 cycles of 1 second at 95°C and 20 seconds at 60°C. Samples were considered negative if the cycle threshold (Ct) was > 40 or if the amplification curve had an abnormal shape.

Each pathogen was tested for using a specific primer/probe set. A tick that was tested positive for a pathogen once was tested at least one more time using a primer/ probe set specific for the same pathogen. A Taqman® probe for detection of B. microti from Teal et al. (2012) that is specific for only this Babesia species was used. A Taqman® probe targeting the msp2 gene of A. phagocytophilum from Courtney, Kostelnik, Zeidner, and Massung (2004) was used with slight modifications. Nested conventional PCR was later used to identify which strain (ha or AP-1) each A. phagocytophilum positive tick was. The Taqman® probe from Courtney et al. (2004) designed to detect 23S rRNA of B. burgdorferi (senso lato) was used with slight changes for optimization. This probe also detects B. miyamotoi, so the method of Dibernado, Cote, Ogden, and Lindsay (2014) was adapted to distinguish tick samples as B. burgdorferi or B. miyamotoi when they that tested positive for the 23S rRNA gene.

An OspA probe specific for the B. burgdorferi was used to confirm B. burgdorferi positive samples and also determine candidates for samples containing B. miyamotoi, which would test negative with this probe. B. miyamotoi contenders were amplified using conventional PCR and the products sequenced by GeneWiz, Inc. South Plainfield, NJ.

Results

Rates of infection for B. burgdorferi (sensu lato), A. phagocytophilum, B. microti, and

B. miyamotoi, were determined for 428 ticks that were collected and tested (Table 1). For tick samples that came up positive for the 23S rRNA gene and negative for the OspA assay, the IGS sequence (16S-23S) and Flagellin sequence specific to B. miyamotoi were amplified using conventional PCR and then sequenced (Bunikis et al., 2004) Of those sequenced so far, one tick was an identical match for B. miyamotoi. Samples positive for the msp2 sequence of A. phagocytophilum by real time PCR were studied further to discriminate between the pathogenic and nonpathogenic strain. For 6 of the 8 positive ticks so far, the 16S rRNA gene was amplified using nested conventional PCR and sequenced (Massung, Priestley, Miller, Mather, & Levin, 2003). All 6 were the AP-1 variant that causes infection in deer, not humans. The 18S rRNA gene of three ticks positive for Babesia microti was amplified and sequenced. The results revealed that two of the ticks carried B. microti and that the third carried B. odocolei, a species that affects elk and not humans.

Table 1: Rates of infection in Lehigh Valley Area ticks as of 07/18/14.

Pathogen N positive/ N tested % Positive

B. burgdorferi (sensu lato) 101/428 23.6%

B. miyamotoi 1/428 0.23%

A. phagocytophilum 8/428 1.9%

B. microti 2/428 0.48%

B. odocoilei 1/428 0.23%

Discussion

The objective of this study was to estimate the prevalence of B. burgdorferi, A. phagocytophilum, B. microti, and B. miyamotoi in deer ticks of the Lehigh Valley in eastern Pennsylvania using sensitive and specific methods. To have high confidence in the accuracy of results that will have clinical significance, much focus was placed on using the most specific methods currently available to determine which ticks carried human pathogens. This required complementing real time PCR assays, which are highly sensitive, with conventional PCR techniques and sequencing of PCR products. The sensitivity of the real time PCR assays is currently being studied to determine approximately how much tick and pathogen DNA can be reliably detected.


B. burgdorferi sensu lato

The 23.6% prevalence of B. burgdorferi in blacklegged ticks in the Lehigh Valley is comparable to the results of Courtney et al. 2003. In their study, deer ticks were collected in 2000 and 2001 in Erie County, located in the northwest corner of Pennsylvania, as well as from Delaware and Chester counties near Lehigh County. Engorged female ticks removed from white-tailed deer in Ridley Creek State Park as well as questing ticks were used in the study. In 2000, 8.8% of the ticks collected in Delaware County were positive for B. burgdorferi. This is a low percentage for an endemic region, but is supported by the low number of cases of Lyme Disease (3.7 cases per 100,000) reported by Delaware County in 2013 (Pennsylvania Department of Health, 2014) Nearby in Chester County, 24.1% of ticks tested positive for B. burgdorferi in 2001. The number of reported cases per 100,000 in 2013 was 159.5, considerably higher than the rate of Delaware County (Pennsylvania Department of Health, 2014). The prevalence of B. burgdorferi found in Lehigh County falls in between that of Delaware and Chester counties; this corresponds to how the rate of 47 cases per 100,000 reported by Lehigh County falls between the numbers of reported cases by Delaware and Chester counties.

In areas where prevalence of B. burgdorferi is more than 20%, use of antibiotic prophylaxis after a tick bite is recommended in guidelines of the Infectious Diseases Society of America (Wormser et al., 2006).

A. phagocytophilum

The overall infection rate in deer ticks of A. phagocytophilum was 1.9%. So far, six of the eight tick samples that were positive for msp2 in real-time PCR were all identified as the AP-1 variant. In Courtney et al. 2003, a similarly low rate of infection (2.5%) of A. phagocytophilum was found in deer ticks in Erie Country in 2001, but all the samples were the ha variant that causes infection in humans. The infection rates in Chester and Delaware counties were higher at 14.8% and 49.6% respectively. While both variants were found among the A. phagocytophilum positive tick samples from both counties, it was especially interesting that over 80% of the engorged female ticks collected in both counties carried the AP-1 variant. A possible source of the high infection rates are the recent blood meals the engorged ticks took from white-tailed deer, a suspected reservoir for the AP-1 variant (Courtney et al. 2003). This result and the sole presence of the ha variant in Erie County lends support to the notion suggested in Courtney et al. 2003 that the AP-1 variant is more abundant where both strains are present. If this is true, it is likely that deer ticks of the Lehigh Valley carry the human variant, but to a lesser degree.

B. microti

Three ticks collected from the same site on the same day tested positive for the 18S rRNA sequence in the real time PCR assay. When they were later sequenced, two ticks carried B. microti and the other carried B. odocolei. In a study done in the Hudson Valley of New York, ticks were pooled for testing and a 6% prevalence of B. microti was found among the deer ticks (Aliota 2014). The probe designed to detect the 18S rRNA gene that was adapted from the Teal et al. (2012) did not detect B. odocolei in their study, but did in ours implying that additional sequencing is necessary to confirm positive results.

B. miyamotoi

B. miyamotoi is a rare, emerging pathogen in the United States and was screened for with part of the method described by Dibernado et al., 2014. One tick was sequenced and confirmed to carry B. miyamotoi.

References

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Comments

Mentor: Luther V. Rhodes III, MD

Department(s)

Research Scholars, Research Scholars - Posters

Document Type

Poster

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