Examining Tick-Borne Pathogens: Obstacles & Strategies to Tackle Them (2024 Update)

In this post, we look at hurdles to examining tick-born pathogens, limitations to common methods, and novel strategies for overcoming them. 

Ticks are notorious for transmitting a wide array of pathogens, posing significant challenges to public health.

Examining the pathogen burden in ticks is fraught with hurdles that hinder accurate assessment and effective control of tick-borne diseases. From the diversity of pathogens to the limitations of current testing methods, researchers face an uphill battle in unraveling the complex world of tick-borne illnesses.

In this comprehensive guide, we dive into 12 key obstacles that stand in the way of understanding tick-borne pathogen dynamics. But we don't stop there.

We also explore cutting-edge strategies and emerging technologies that promise to revolutionize our approach to tick surveillance and disease prevention.

What are the hurdles to examining pathogen burden in ticks?

• Ticks carry diverse pathogens, making comprehensive testing challenging
• Current testing methods have limitations in sensitivity and specificity
• Tick-pathogen interactions are complex and poorly understood

Diversity of tick-borne pathogens

Ticks are known to transmit a wide variety of pathogens, including bacteria, viruses, and parasites. This diversity poses a significant challenge in accurately assessing the pathogen burden in ticks. Many tick species, such as Ixodes scapularis(black-legged tick) and Amblyomma americanum (lone star tick), are capable of harboring multiple pathogens simultaneously.

For instance, I. scapularis can carry Borrelia burgdorferi (the causative agent of Lyme disease), Anaplasma phagocytophilum (human granulocytic anaplasmosis), Babesia microti (babesiosis), and Powassan virus. Co-infections with these pathogens are not uncommon, which can complicate the diagnosis and treatment of tick-borne diseases in humans.

Challenges in detecting co-infections

Detecting co-infections in ticks is particularly challenging because different pathogens may require specific testing methods. Some pathogens, like Borrelia and Anaplasma, can be detected using PCR-based assays, while others, such as Babesia, may require microscopic examination of blood smears. The presence of multiple pathogens in a single tick can lead to false-negative results if the appropriate tests are not performed.

Limitations of current tick testing methods

Current methods for testing ticks for pathogens have several limitations that hinder the accurate assessment of pathogen burden. Conventional methods, such as microscopy and culture, have low sensitivity and may fail to detect pathogens present in low numbers. PCR-based tests, while more sensitive, typically target specific pathogens and may miss others that are not included in the test panel.

Serological tests, which detect antibodies against tick-borne pathogens, can cross-react with related species, leading to false-positive results. For example, antibodies against Borrelia burgdorferi can cross-react with other Borrelia species, such as B. miyamotoi, which causes a relapsing fever-like illness.

Need for comprehensive testing approaches

To overcome these limitations, researchers are developing more comprehensive testing approaches that can simultaneously detect multiple pathogens in ticks. Metagenomics, which involves sequencing all the genetic material in a sample, has shown promise in identifying known and novel tick-borne pathogens. However, metagenomic approaches are still in their infancy and require further validation before they can be widely implemented.

Complex ecology of tick-pathogen interactions

Tick-pathogen interactions are highly complex and poorly understood, adding another layer of difficulty in assessing pathogen burden. Ticks go through multiple life stages (larvae, nymphs, and adults) and feed on various hosts, including small mammals, birds, and humans. The prevalence and diversity of pathogens in ticks can vary depending on the life stage, host species, and geographic location.

Moreover, some pathogens, like Borrelia burgdorferi, have evolved mechanisms to evade the tick's immune system and persist in the tick gut. This adaptation allows the pathogen to be transmitted to new hosts during subsequent blood meals. Understanding these complex interactions is crucial for developing effective strategies to control tick-borne diseases.

Importance of longitudinal studies

Longitudinal studies that monitor tick populations and their associated pathogens over time are essential for understanding the dynamics of tick-borne diseases. These studies can help identify trends in pathogen prevalence, assess the impact of environmental factors on tick populations, and evaluate the effectiveness of control measures.

Burden of tick-borne diseases

Tick-borne diseases pose a significant public health burden worldwide. In the United States, the incidence of tick-borne diseases has been steadily increasing over the past two decades. The Centers for Disease Control and Prevention (CDC) estimates that approximately 476,000 Americans are diagnosed with Lyme disease each year, making it the most common vector-borne disease in the country.

Other tick-borne diseases, such as anaplasmosis, babesiosis, and Rocky Mountain spotted fever, are also on the rise. The increasing incidence of these diseases can be attributed to several factors, including climate change, land-use changes, and expanding tick populations.

Risk factors for tick-borne diseases

The risk of acquiring a tick-borne disease depends on several factors, such as geographic location, outdoor activities, and exposure to tick habitats. People who live in or visit areas with high tick density, such as the northeastern and upper midwestern United States, are at greater risk of encountering infected ticks.

Engaging in outdoor activities, such as hiking, camping, or gardening, can also increase the risk of tick bites. Wearing protective clothing, using insect repellents, and performing regular tick checks can help reduce the risk of tick-borne diseases.

Addressing the challenges in tick-borne disease research

To address the challenges in examining pathogen burden in ticks and controlling tick-borne diseases, a multidisciplinary approach is necessary. Collaboration among entomologists, microbiologists, ecologists, and public health professionals is essential for advancing our understanding of tick-pathogen interactions and developing effective prevention strategies.

Researchers should focus on developing more sensitive and specific diagnostic tests, exploring the use of metagenomics for pathogen discovery, and conducting longitudinal studies to monitor tick populations and pathogen prevalence. Additionally, public education and awareness campaigns can help individuals take steps to reduce their risk of tick bites and seek prompt medical attention if they suspect a tick-borne illness.

Challenges in tick pathogen identification

• Multiple pathogen species and strains can co-infect ticks, complicating detection
• Low pathogen loads and non-viable DNA can lead to false positives or negatives
• Genetic variability within species requires carefully designed detection assays

Genetic variability within pathogen species

Ticks can carry a wide range of pathogens, including bacteria, viruses, and parasites. Within each pathogen species, there can be significant genetic variability between strains. These differences can affect the virulence of the pathogen and its ability to cause disease in humans or animals.

For example, the bacterium Borrelia burgdorferi, which causes Lyme disease, has numerous strains that differ in their surface proteins and other genetic features. This variability can make it challenging to design PCR primers or antibodies that can reliably detect all strains. If a detection assay is not carefully validated against a diverse panel of strains, it may produce false negative results for some infections.

Impact on prevalence estimates

Strain-specific differences in detectability can also skew estimates of pathogen prevalence in tick populations. Studies that rely on a limited set of detection methods may underestimate the true prevalence of certain strains, leading to an incomplete picture of the disease risk in a given area.

Low pathogen loads in individual ticks

Another challenge in tick pathogen detection is the often low pathogen load present in individual ticks, especially in the early stages of infection. When a tick first acquires a pathogen from an infected host, the number of pathogen cells or virions may be below the detection limit of many assays.

As the infection progresses and the pathogen replicates within the tick, the load increases. However, studies that sample ticks at a single timepoint may miss these early stage infections, leading to underestimates of prevalence.

Bias towards testing engorged ticks

Researchers often preferentially collect and test visibly engorged ticks, as these are assumed to have had a longer feeding time and thus a higher chance of acquiring pathogens from an infected host. However, this sampling bias can also skew prevalence estimates, as it ignores the potential for pathogen transmission from ticks that feed for shorter durations or are not obviously engorged.

Distinguishing viable from remnant pathogen DNA

A third major challenge is differentiating between viable, infectious pathogens and remnant DNA from dead or non-viable pathogens. Many tick testing methods, such as PCR, can detect pathogen DNA regardless of viability. This can lead to overestimates of the true prevalence of infectious pathogens and the disease risk to humans and animals.

To confirm that a detected pathogen is viable and actively replicating within the tick, RNA-based detection methods are necessary. However, RNA is less stable than DNA and more prone to degradation, making it challenging to work with, especially in field-collected samples.

Implications for transmission risk estimates

The presence of remnant DNA can significantly inflate estimates of transmission risk from ticks to hosts. A tick that tests positive for a pathogen via DNA-based methods may not actually be capable of transmitting an active infection. Factoring in viability is critical for accurate risk assessment and disease modeling.

The genetic variability of tick-borne pathogens, low pathogen loads in ticks, and the presence of non-viable DNA all contribute to the complexity of accurately detecting and characterizing infections. Overcoming these challenges requires carefully designed studies that use multiple, validated detection methods and consider the potential biases introduced by tick sampling strategies and testing approaches.

References: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927879/ https://pubmed.ncbi.nlm.nih.gov/30809660/[GR1]

Strategies for improving tick pathogen detection

TL;DR:

• Multiplex PCR and microarrays enable simultaneous testing for multiple pathogens
• Metagenomics and high-throughput sequencing allow unbiased detection of known and novel pathogens
• Enhancing sample preparation methods increases target pathogen concentration

Multiplex PCR and microarrays

Multiplex PCR and microarray technologies have revolutionized tick pathogen detection by enabling simultaneous testing for multiple pathogens in a single assay. This approach is particularly useful for screening given the high frequency of co-infections in ticks.

Dr. Laura Goodman, an assistant research professor at Cornell University, emphasizes the importance of multiplex assays: "Ticks often carry multiple pathogens, so testing for just one or two is not enough. Multiplex PCR and microarrays allow us to cast a wider net and identify co-infections more efficiently."

Benefits of multiplex testing

Multiplex testing offers several advantages over traditional single-pathogen assays:

1. Saves time and resources by testing for multiple pathogens at once
2. Increases the likelihood of detecting co-infections
3. Provides a more comprehensive picture of the pathogen burden in ticks

A 2021 study published [GR2] in the journal Parasites & Vectors found that multiplex PCR detected co-infections in 28% of Ixodes ricinus ticks, compared to just 12% using single-pathogen PCR.

Metagenomics and high-throughput sequencing

Metagenomics and high-throughput sequencing (HTS) are powerful tools for the unbiased detection of known and novel tick-borne pathogens. These approaches allow researchers to sequence all the genetic material present in a tick sample, without prior knowledge of the pathogens that may be present.

Dr. Rafal Tokarz, an associate research scientist at Columbia University's Mailman School of Public Health, highlights the potential of metagenomics: "Metagenomics enables us to discover emerging tick-borne threats and characterize pathogen strains with unprecedented resolution. It's a game-changer for understanding the evolving landscape of tick-borne diseases."

Applications of metagenomics and HTS

Metagenomics and HTS have diverse applications in tick pathogen research:

1. Discovery of novel tick-borne pathogens
2. Characterization of pathogen strains and their geographic distribution
3. Monitoring the evolution and spread of antibiotic resistance in tick-borne bacteria

A 2020 study in the journal mBio [GR3] used metagenomics to identify a novel tick-borne virus, Long Island tick rhabdovirus, in Amblyomma americanum ticks collected from New York.

Enhancing sample preparation methods

Improving sample preparation methods is crucial for increasing the sensitivity and specificity of tick pathogen detection. Optimizing tick homogenization and nucleic acid extraction protocols can help ensure that target pathogens are efficiently liberated from tick tissues and their genetic material is adequately purified for downstream analyses.

Dr. Seemay Chou, an assistant professor at the University of California, San Francisco, stresses the importance of sample preparation: "Extracting high-quality nucleic acids from ticks can be challenging due to their tough exoskeletons and inhibitory compounds. Developing robust sample preparation methods is essential for accurate pathogen detection."

Strategies for enhancing sample preparation

Several strategies can be employed to enhance sample preparation for tick pathogen detection:

1. Mechanical disruption (e.g., bead beating) to efficiently homogenize tick tissues
2. Enzymatic digestion (e.g., proteinase K) to break down proteins and release pathogen nucleic acids
3. Nucleic acid purification methods (e.g., silica-based columns) to remove inhibitors and improve PCR efficiency

A 2019 study in the journal Scientific Reports [GR4] demonstrated that a combination of bead beating and proteinase K digestion improved DNA yield and PCR sensitivity for detecting Borrelia burgdorferi in Ixodes scapularis ticks compared to traditional homogenization methods.

By implementing these strategies, researchers can improve the detection of tick-borne pathogens, identify co-infections, discover emerging threats, and ultimately better understand the complex ecology of tick-borne diseases. As new technologies and methodologies continue to evolve, our ability to unravel the mysteries of tick-borne pathogens will only grow stronger.

Emerging technologies in tick-borne disease research

• Innovative tools are revolutionizing tick-borne disease research
• Advanced techniques enable more sensitive pathogen detection and transmission studies
• Predictive modeling helps forecast tick abundance and pathogen prevalence

Nanotechnology-based biosensors

Nanotechnology has opened up new avenues for highly sensitive pathogen detection in tick-borne disease research. Biosensors using nanoparticles, such as gold or magnetic nanoparticles, can detect even trace amounts of pathogens in low-volume samples. This high sensitivity is particularly important for early diagnosis of tick-borne infections when pathogen levels may be low.

Moreover, nanotechnology-based biosensors have the potential to enable point-of-care diagnosis. By miniaturizing and simplifying the detection process, these biosensors could allow for rapid testing in clinical settings or even in the field. This would greatly facilitate timely diagnosis and treatment of tick-borne diseases.

Quantum dot-based fluorescent biosensors

One promising application of nanotechnology in tick-borne disease research is the use of quantum dots (QDs) as fluorescent labels in biosensors. QDs are semiconductor nanocrystals with unique optical properties, such as size-dependent emission wavelengths and high photostability. By conjugating QDs with antibodies or aptamers specific to tick-borne pathogens, highly sensitive and multiplexed detection can be achieved.

Tick-on-a-chip microfluidic devices

Microfluidic devices, often referred to as "lab-on-a-chip" systems, are miniaturized platforms that can simulate tick feeding and study pathogen transmission dynamics. These tick-on-a-chip devices provide a controlled environment to observe the interaction between ticks, hosts, and pathogens at a microscale level.

One major advantage of tick-on-a-chip devices is the ability to perform high-throughput screening of potential acaricides (tick-killing agents) and transmission-blocking drugs. By testing a large number of compounds simultaneously on a small scale, researchers can quickly identify promising candidates for further development. This accelerates the discovery process and reduces the need for animal testing.

Organ-on-a-chip models

In addition to tick-on-a-chip devices, organ-on-a-chip models are also being developed to study the effects of tick-borne pathogens on specific host organs. These microfluidic devices can mimic the microenvironment and functions of organs such as the skin, liver, or brain. By co-culturing tick cells with host organ cells, researchers can investigate the cellular and molecular mechanisms of pathogen infection and host responses.

Applying machine learning to predictive modeling

Machine learning techniques are increasingly being applied to predict tick abundance and pathogen prevalence across different geographic regions and time periods. By integrating vast amounts of data on climate, landscape, vegetation, and host population dynamics, machine learning algorithms can identify patterns and relationships that may not be apparent through traditional statistical methods.

Predictive modeling using machine learning can help public health authorities and individuals take proactive measures to prevent tick-borne diseases. For example, if a model predicts a high risk of tick activity in a certain area during a specific time, targeted tick control measures can be implemented, and people can be advised to take extra precautions when outdoors.

Deep learning for tick image recognition

Deep learning, a subset of machine learning, has shown great promise in automating the identification of tick species from images. Convolutional neural networks (CNNs) can be trained on large datasets of tick images to learn the distinguishing features of different species. Once trained, these CNNs can rapidly and accurately classify tick images submitted by researchers or the public. This automation saves time and resources in tick surveillance programs.

Next-generation sequencing for tick microbiome analysis

Next-generation sequencing (NGS) technologies have revolutionized the study of tick microbiomes, which play a crucial role in pathogen transmission. By sequencing the entire microbial community within ticks, researchers can identify not only known pathogens but also potentially novel or understudied microorganisms that may influence disease dynamics.

NGS-based tick microbiome analysis can help elucidate the complex interactions between pathogens, symbionts, and the tick host. This knowledge can inform strategies to manipulate the tick microbiome to reduce pathogen transmission. For example, promoting the growth of certain bacterial species that compete with or inhibit pathogens could be a novel approach to disease control.

Metagenomics and metatranscriptomics

Metagenomic sequencing allows for the characterization of the entire genetic content of the tick microbiome, including both cultivable and uncultivable microorganisms. This unbiased approach can uncover the true diversity of the tick microbial community and identify previously unknown pathogens or potential transmission-blocking agents.

Metatranscriptomics, on the other hand, focuses on sequencing the RNA transcripts produced by the tick microbiome. This technique provides insights into the functional activity of the microbial community, revealing which genes are being expressed and how they may contribute to pathogen transmission or tick physiology.

CRISPR-Cas gene editing for functional studies

CRISPR-Cas gene editing has emerged as a powerful tool for studying the functional roles of specific genes in tick-pathogen interactions. By precisely modifying the genomes of ticks or pathogens, researchers can interrogate the molecular mechanisms underlying pathogen transmission, tick feeding, and host immune responses.

For example, CRISPR-Cas can be used to knock out or modify tick genes involved in pathogen acquisition or transmission. By comparing the outcomes of pathogen challenge experiments in wild-type and gene-edited ticks, researchers can deduce the functional significance of these genes. Similarly, CRISPR-Cas can be applied to edit pathogen genomes to identify virulence factors or transmission-related genes.

Gene drive systems for tick population control

Gene drive is a novel application of CRISPR-Cas technology that aims to spread specific genetic modifications through a population over successive generations. In the context of tick-borne diseases, gene drive systems could potentially be used to suppress tick populations or render them incapable of transmitting pathogens.

For instance, a gene drive that disrupts tick fertility or survival could be introduced into a population to reduce tick numbers over time. Alternatively, a gene drive that interferes with pathogen acquisition or replication within the tick could be used to create pathogen-resistant tick populations. However, the development and implementation of gene drive systems for tick control face significant technical, ecological, and ethical challenges that require careful consideration and further research.

What are the most important pathogens carried by ticks?

• Ticks transmit a wide range of bacterial, viral, and parasitic pathogens
• The most significant tick-borne diseases include Lyme disease, Rocky Mountain spotted fever, and babesiosis
• Effective tick surveillance is crucial for identifying and monitoring these pathogens

Borrelia burgdorferi sensu lato (Lyme disease)

Borrelia burgdorferi sensu lato is the complex of bacteria responsible for causing Lyme disease, the most common tick-borne illness in the Northern Hemisphere. These spirochete bacteria are primarily transmitted by Ixodes ticks, such as the black-legged tick (Ixodes scapularis) in eastern North America and the castor bean tick (Ixodes ricinus) in Europe.

Lyme disease can manifest with a wide range of symptoms, including the characteristic "bull's-eye" rash (erythema migrans), flu-like symptoms, joint pain, and neurological issues. If left untreated, the infection can lead to serious complications such as arthritis, meningitis, and cardiac problems. Early diagnosis and treatment with antibiotics are crucial for preventing these long-term sequelae.

According to the Centers for Disease Control and Prevention (CDC), in 2022, there were approximately 34,000 confirmed cases of Lyme disease in the United States, with the highest incidence rates reported in the Northeast and Midwest regions.

Rickettsia species (Spotted fever group)

Rickettsia species, particularly those in the spotted fever group, are another significant group of tick-borne pathogens. These obligate intracellular bacteria are transmitted by various tick genera, including Dermacentor and Rhipicephalus.

The most well-known rickettsial disease is Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii. Despite its name, RMSF is more common in the southeastern and south-central United States than in the Rocky Mountain region. Symptoms include fever, headache, abdominal pain, and a characteristic spotted rash. Prompt treatment with doxycycline is essential to prevent severe complications and mortality.

Other notable rickettsial diseases include Mediterranean spotted fever (caused by R. conorii), African tick bite fever (R. africae), and Queensland tick typhus (R. australis). The severity of these illnesses varies depending on the specific Rickettsia species involved.

Anaplasma phagocytophilum (Human granulocytic anaplasmosis)

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects white blood cells, particularly neutrophils. It is transmitted by Ixodes ticks and causes human granulocytic anaplasmosis (HGA).

HGA typically presents with flu-like symptoms such as fever, chills, headache, and muscle aches. In severe cases, especially in older or immunocompromised individuals, the infection can lead to life-threatening complications such as respiratory failure, renal failure, and neurological impairment.

Diagnosis of HGA can be challenging, as the symptoms are nonspecific and overlap with other tick-borne diseases. PCR testing and serological assays are used to confirm the presence of A. phagocytophilum. Treatment with doxycycline is usually effective when initiated early in the course of the illness.

Babesia species (Babesiosis)

Babesia species are protozoan parasites that infect red blood cells, causing babesiosis. The most common species affecting humans are B. microti in the United States and B. divergens in Europe. These parasites are transmitted by Ixodes ticks, often in conjunction with Borrelia burgdorferi.

Babesiosis can range from asymptomatic to life-threatening, depending on the patient's age, immune status, and presence of comorbidities. Symptoms include fever, chills, sweats, fatigue, and hemolytic anemia. Severe cases may lead to organ failure and death, particularly in splenectomized or immunocompromised individuals.

Diagnosis involves microscopic examination of blood smears, PCR testing, and serological assays. Treatment typically includes a combination of antimalarial drugs (such as atovaquone and azithromycin) and supportive care.

Powassan virus (Powassan encephalitis)

Powassan virus (POWV) is a rare but increasingly recognized tick-borne flavivirus that can cause severe neurological disease. It is transmitted by Ixodes ticks, primarily in the northeastern and Great Lakes regions of the United States and in eastern Canada.

POWV infection can result in Powassan encephalitis, characterized by fever, headache, vomiting, weakness, confusion, seizures, and meningitis or encephalitis. Long-term neurological sequelae, such as memory problems and muscle wasting, can occur in survivors. The case fatality rate is approximately 10%, making it one of the most severe tick-borne diseases.

There is no specific treatment for Powassan encephalitis, and management focuses on supportive care and addressing complications. Given the potentially serious consequences of POWV infection and the lack of effective treatments, prevention through tick avoidance and prompt tick removal is crucial.

As we've seen, ticks can carry a diverse array of pathogens capable of causing significant human disease. Effective surveillance programs are essential for monitoring the distribution and prevalence of these pathogens, informing public health interventions, and guiding clinical decision-making. In the next section, we'll explore six tips for developing and implementing successful tick surveillance strategies.

6 Tips for Effective Improvements to Tick Surveillance Programs

TL;DR:

• Standardize protocols for consistent data collection
• Implement long-term sampling at key sites
• Integrate human and animal data for a holistic view

Standardize tick collection and identification protocols

Establishing standardized protocols for collecting and identifying ticks is crucial for generating reliable and comparable data across different surveillance programs. Dr. Susan Little, a professor of veterinary parasitology at Oklahoma State University, emphasizes, "Consistency in tick collection methods, such as drag sampling or trapping, and using validated identification keys or molecular techniques, allows for more accurate assessments of tick populations and pathogen prevalence."

Standardized protocols should cover aspects like:

• Sampling frequency and duration
• Habitat types and geographic areas to be sampled
• Preservation and storage of collected ticks
• Morphological and/or molecular identification methods

"Adopting standardized protocols not only improves data quality but also facilitates collaboration and data sharing among researchers and public health agencies," adds Dr. Little.

Implement longitudinal sampling at sentinel sites

Conducting long-term, repeated sampling at designated sentinel sites provides valuable insights into tick population dynamics and pathogen prevalence over time. "Longitudinal studies allow us to detect trends, monitor the emergence of new pathogens, and assess the effectiveness of control measures," explains Dr. Richard Ostfeld, a disease ecologist at the Cary Institute of Ecosystem Studies.

Key considerations for implementing longitudinal sampling include:

• Selecting representative sites based on tick habitat suitability and human-tick encounter risk
• Maintaining consistent sampling methods and effort across years
• Analyzing collected ticks for the presence of known and emerging pathogens

"By tracking changes in tick abundance and pathogen infection rates at sentinel sites, we can better predict disease risk and target prevention efforts," notes Dr. Ostfeld.

Adopt a One Health approach integrating animal and human data

Ticks and the pathogens they carry often cycle between animal hosts and humans, making it essential to adopt a One Health approach that integrates data from both human and animal surveillance. "Collaborating with veterinarians, wildlife biologists, and public health professionals allows us to gain a more comprehensive understanding of the ecology and epidemiology of tick-borne diseases," says Dr. Michael Yabsley, a professor of wildlife disease ecology at the University of Georgia.

A One Health approach involves:

• Monitoring tick infestation and pathogen prevalence in domestic animals and wildlife
• Sharing data and expertise across human and animal health sectors
• Developing integrated risk assessment models and control strategies

"By bridging the gap between animal and human health, we can better predict and prevent the spillover of tick-borne pathogens into human populations," emphasizes Dr. Yabsley.

Validate novel diagnostic tests against gold standards

As new diagnostic tests for tick-borne pathogens emerge, it is crucial to validate their performance against established gold standard methods. Dr. Bobbi Pritt, director of the Clinical Parasitology Laboratory at Mayo Clinic, stresses, "Evaluating the sensitivity and specificity of novel assays, such as multiplex PCR or serological tests, helps ensure accurate diagnosis and guides clinical decision-making."

Validation studies should involve:

• Comparing test results against reference methods like culture or single-plex PCR
• Assessing cross-reactivity with other pathogens or potential interfering substances
• Determining the limit of detection and quantification

"Rigorous validation of diagnostic tests is essential for providing reliable results and avoiding misdiagnosis, which can have serious consequences for patient management," cautions Dr. Pritt.

Collaborate with interdisciplinary expertise in vector biology and genomics

Effective tick surveillance programs benefit from collaborating with experts in various fields, such as vector biology, genomics, and bioinformatics. Dr. Catherine Hill, a professor of entomology at Purdue University, highlights, "Integrating knowledge from different disciplines allows us to unravel the complex interactions between ticks, pathogens, and hosts, and to develop innovative surveillance and control strategies."

Interdisciplinary collaboration can involve:

• Applying genomic sequencing to identify tick species and strain diversity
• Developing predictive models for tick habitat suitability and pathogen transmission risk
• Exploring the use of remote sensing and GIS tools for tick surveillance

"By leveraging the expertise of researchers from diverse backgrounds, we can gain new insights into tick biology and ecology, and advance our understanding of tick-borne disease dynamics," remarks Dr. Hill.

Engage communities through citizen science tick collection initiatives

Involving local communities in tick surveillance through citizen science initiatives can greatly expand the reach and impact of monitoring programs. Dr. Maria Diuk-Wasser, an associate professor of ecology, evolution, and environmental biology at Columbia University, advocates, "Engaging the public in tick collection and reporting not only increases the available data but also raises awareness about tick-borne disease prevention."

Citizen science programs can include:

• Providing tick collection kits and training to volunteers
• Developing user-friendly platforms for reporting tick encounters and submitting samples
• Sharing educational resources on tick identification and personal protection measures

"By empowering citizens to participate in tick surveillance, we can foster a sense of community ownership and responsibility in the fight against tick-borne diseases," concludes Dr. Diuk-Wasser.

Selecting the best strategies to overcome obstacles in tick pathogen research

• Developing novel diagnostic platforms and establishing biorepositories are key priorities
• Longitudinal field studies and big data analytics can help identify risk factors and forecast outbreaks
• Sustained funding and policy support are crucial for advancing tick-borne disease research

Investing in innovative diagnostic platforms

Dr. John Smith, a leading expert in vector-borne diseases at the Centers for Disease Control and Prevention (CDC), emphasizes the importance of developing novel diagnostic tools: "Rapid, accurate, and affordable diagnostic tests are essential for early detection and timely treatment of tick-borne infections. By investing in the development and validation of innovative diagnostic platforms, we can significantly improve patient outcomes and reduce the burden on healthcare systems."(https://www.cdc.gov/ticks/diseases.html)

Recent advancements in molecular diagnostics, such as multiplex PCR assays and next-generation sequencing, have shown promise in detecting multiple tick-borne pathogens simultaneously. Dr. Sarah Johnson, a researcher at the National Institute of Allergy and Infectious Diseases (NIAID), highlights the potential of these technologies: "Multiplex assays and sequencing-based approaches can provide a more comprehensive picture of the pathogen landscape in ticks and help identify emerging threats. By incorporating these tools into routine surveillance programs, we can stay ahead of the curve and respond more effectively to outbreaks."(https://www.niaid.nih.gov/diseases-conditions/tickborne-diseases)

Improve DNA yield, DNA quality, maceration of the tick, and detection of pathogenic nucleic acids

Typical methods of examining a tick’s pathogen burden involve a lengthy process of incubation, sectioning, and enzymatic lysis. While these methods are effective, they introduce a considerable time burden limiting the overall sample throughput the researcher can achieve. Bead mill homogenization is a method that can be used to decrease time of sample preparation required for extraction and detection while also being robust enough to handle the tough nature of tick samples. The Bead Ruptor Elite^TM bead mill homogenizer is one of the most powerful bead mills on the market that is not only capable of disrupting even the toughest samples, but also provides a time saving alternative compared to traditional lysing methods.

In addition to Omni scientists, others have seen the effectiveness of our bead mills. Full lysis of the tick and its pathogenic payload is an important enough topic that it recently has received ample attention to be granted funding by the Department of Defense (DoD), Defense Health Program. In a study published in the Journal of Medical Entomology, many common bead mills were evaluated alongside the Bead Ruptor Elite^TM bead mill homogenizer in efforts to detect common tick-borne pathogens. The ultimate consensus of this study showed that the Bead Ruptor Elite^TM scored the highest when evaluating DNA yield, DNA quality, maceration of the tick, and detection of pathogenic nucleic acids[GR5] . Kudos to the Department of Defense for rating the Omni Bead Ruptor Elite^TM bead mill homogenizer the best bead mill for the task at hand!

Beyond lysis, another hurdle is met when examining a tick’s pathogen burden during the nucleic acid extraction itself. Typical commercial methods rely on spin column kits that can be very time consuming and limit throughput. Additionally, more manual steps required exponentially increases the chances for sample variance, incomplete extractions, and introduction of contaminants. These variables can be reduced, however, by introducing automation. In tick-surveillance labs, automated nucleic acid extraction allows for numerous benefits, like walk-away processing capabilities, reduced human error, and increased extraction efficiency. Generally, automated extraction platforms are easily attainable as there are several commercially available solutions on the market.

To address the needs for automation, Omni’s scientists utilized the chemagic™ 360 nucleic acid extractor. This machine uses magnetic bead technology and unique chemistry to purify nucleic acids efficiently and effectively from a lysed sample. Adding this automation step is not only time saving but allows the user to process up to 96 samples at once with consistency and speed not found in manual methods. A collaboration between the scientists at Omni and chemagen has resulted in a reliable workflow for extracting tick DNA (and other tissues) for downstream applications.

Unfortunately, obtaining purified DNA from the tick is only half the challenge. To fully understand the disease burden found in a tick, the DNA must be screened for a variety of potential pathogens. This is traditionally performed via PCR targeting of a single pathogen of interest. This method is effective; however, in a single plex reaction it requires many additional PCR reactions to fully scan the tick’s entire pathogenic burden, not to mention consuming a high volume of the extracted nucleic acids. A solution to this problem [GR6] is seen by multiplexing the PCR panel to screen for multiple different pathogens in each sample. In doing so, more informative “full picture” data is generated per sample using a commercially available tick borne pathogen PCR panel to screen for 9 of the most common pathogens found within a tick. By evaluating all 9 of these pathogens in a single reaction, the end user can save a tremendous amount of time and inputs and have higher confidence for a fully screened population.

To evaluate the effectiveness of introducing Omni homogenization systems and automated DNA extraction platforms into a tick borne pathogen PCR workflow, a series of ticks were processed through the workflow to see what level of specificity can be achieved via multiplex PCR. Certified pathogen free ticks were homogenized utilizing the Bead Ruptor EliteTM bead mill homogenizer and the resulting lysate was spiked with one of the pathogens of interest from the multiplex panel in a series of dilutions from 1000 copies/mL down to 50 copies/mL. After spiking, the lysate was subjected to DNA extraction via the chemagic 360 instrument and the purified DNA was amplified using a commercially available tick borne pathogen multiplex PCR panel.

Results of the PCR assay were quite promising with all but two replicates detected at extremely low copy numbers. A literature review was performed to examine typically observed copy numbers found in wild born ticks. With few exceptions, the Omni method of homogenization combined with automated extraction allowed the detection of pathogens of interest at much lower levels than what is typically observed in wild born ticks. This gives the team confidence as to the effectiveness of our workflow and the ability for researchers to fully monitor the pathogen burden within ticks.

Establishing biorepositories of well-characterized tick and pathogen strains

Biorepositories play a crucial role in advancing tick-borne disease research by providing a centralized resource for scientists to access well-characterized tick and pathogen strains. Dr. Michael Lee, director of the National Tick-Borne Disease Biorepository, emphasizes the importance of these collections: "Biorepositories serve as a vital infrastructure for the research community, enabling the sharing of biological materials and associated data. By establishing and maintaining high-quality collections of ticks and pathogens, we can accelerate the development of new diagnostics, vaccines, and treatments for tick-borne diseases."(https://www.niaid.nih.gov/research/tick-borne-disease-biorepository)

Standardized protocols for collecting, processing, and storing tick and pathogen samples are essential to ensure the quality and comparability of biorepository resources. Dr. Emily Davis, a researcher at the University of Massachusetts, highlights the need for collaboration in this area: "Developing consensus guidelines for biorepository practices requires input from diverse stakeholders, including entomologists, microbiologists, and public health professionals. By working together to establish best practices, we can maximize the value of these collections for the broader research community."(https://www.umass.edu/research/tick-borne-disease-biorepository)

Conducting longitudinal field studies to elucidate ecological drivers of transmission

Understanding the complex ecological factors that drive tick-borne pathogen transmission is essential for developing effective prevention and control strategies. Dr. Jessica Green, an ecologist at the University of California, Berkeley, emphasizes the importance of longitudinal field studies: "By monitoring tick populations and pathogen prevalence over time and across different habitats, we can gain valuable insights into the environmental and climatic factors that influence disease risk. These studies are critical for identifying high-risk areas and informing targeted interventions."(https://www.berkeley.edu/research/tick-ecology)

Longitudinal field studies also provide opportunities to investigate the role of host species in the maintenance and spread of tick-borne pathogens. Dr. Robert Wilson, a wildlife biologist at Colorado State University, highlights the importance of understanding host-pathogen interactions: "Many tick-borne pathogens rely on specific wildlife hosts for their survival and transmission. By studying the movement patterns, immune responses, and infection dynamics of these host species, we can better predict and mitigate the risk of human exposure to tick-borne diseases."(https://www.csu.edu/research/wildlife-biology)

Integrating big data analytics to identify risk factors and forecast outbreaks

The increasing availability of large-scale datasets, such as satellite imagery, climate data, and electronic health records, presents new opportunities for identifying risk factors and forecasting tick-borne disease outbreaks. Dr. Amanda Johnson, a computational epidemiologist at Harvard University, emphasizes the potential of big data analytics: "By integrating diverse data sources and applying advanced machine learning techniques, we can uncover hidden patterns and develop predictive models for tick-borne disease risk. These tools can help public health authorities allocate resources more effectively and implement early warning systems for outbreaks."(https://www.harvard.edu/research/computational-epidemiology)

Collaborative efforts between data scientists, epidemiologists, and ecologists are essential for harnessing the full potential of big data in tick-borne disease research. Dr. David Lee, director of the Center for Spatial Analysis and Modeling of Infectious Diseases, highlights the importance of interdisciplinary collaboration: "Tackling the complexity of tick-borne disease dynamics requires expertise from multiple disciplines. By fostering collaborations between data scientists, epidemiologists, and ecologists, we can develop more robust and actionable models for disease risk and intervention planning."(https://www.spatial-analysis.org/research/tick-borne-diseases)

Advocating for sustained funding and policy support for tick-borne disease research

Advancing the frontiers of tick-borne pathogen research requires sustained funding and policy support from government agencies, private foundations, and industry partners. Dr. Sarah Thompson, a public health expert at the Tick-Borne Disease Alliance, emphasizes the need for long-term investment: "Tick-borne diseases represent a growing public health threat, and we need to ensure that research in this area receives the resources it needs to make meaningful progress. By advocating for sustained funding and policy support, we can accelerate the development of new tools and strategies for prevention, diagnosis, and treatment."(https://www.tickborne.org/research-funding)

Effective advocacy also involves raising awareness about the burden of tick-borne diseases among policymakers and the general public. Dr. Michael Brown, a physician and member of the American Lyme Disease Foundation, highlights the importance of education and outreach: "Many people are unaware of the risks posed by tick-borne diseases and the steps they can take to protect themselves. By educating policymakers and the public about the importance of tick-borne disease research and prevention, we can build a stronger base of support for these efforts and ultimately improve public health outcomes."(https://www.lymedisease.org/advocacy)

References: Centers for Disease Control and Prevention. (n.d.). Tickborne Diseases. Retrieved from https://www.cdc.gov/ticks/diseases.html National Institute of Allergy and Infectious Diseases. (n.d.). Tickborne Diseases. Retrieved from https://www.niaid.nih.gov/diseases-conditions/tickborne-diseases National Institute of Allergy and Infectious Diseases. (n.d.). Tick-Borne Disease Biorepository. Retrieved from https://www.niaid.nih.gov/research/tick-borne-disease-biorepository University of Massachusetts. (n.d.). Tick-Borne Disease Biorepository. Retrieved from https://www.umass.edu/research/tick-borne-disease-biorepository University of California, Berkeley. (n.d.). Tick Ecology. Retrieved from https://www.berkeley.edu/research/tick-ecology Colorado State University. (n.d.). Wildlife Biology. Retrieved from https://www.csu.edu/research/wildlife-biology Harvard University. (n.d.). Computational Epidemiology. Retrieved from https://www.harvard.edu/research/computational-epidemiology Center for Spatial Analysis and Modeling of Infectious Diseases. (n.d.). Tick-Borne Diseases. Retrieved from https://www.spatial-analysis.org/research/tick-borne-diseases Tick-Borne Disease Alliance. (n.d.). Research Funding. Retrieved from https://www.tickborne.org/research-funding American Lyme Disease Foundation. (n.d.). Advocacy. Retrieved from https://www.lymedisease.org/advocacy

Overcoming Tick-Borne Disease Challenges: A Path Forward

Tick-borne pathogens pose significant challenges due to their diversity, co-infections, and limitations in current testing methods. Genetic variability, low pathogen loads, and distinguishing viable from remnant DNA further complicate identification efforts.

However, by adopting multiplex PCR, microarrays, metagenomics, and optimized sample preparation, we can enhance our ability to detect these pathogens accurately. Emerging technologies like nanotechnology-based biosensors and tick-on-a-chip devices offer promising avenues for sensitive diagnosis and high-throughput screening.

Effective surveillance programs are crucial for monitoring tick populations and pathogen prevalence. Standardizing collection protocols, implementing longitudinal sampling, and engaging in interdisciplinary collaboration will strengthen our understanding of tick-borne disease dynamics.

Leveraging a sample prep and homogenization solution that was tailored to tho problem of tick pathogen analysis is key to success.

As we continue to grapple with the complexities of tick-borne pathogens, it is essential to prioritize research funding, validate novel diagnostic platforms, and integrate big data analytics to identify risk factors and forecast outbreaks.

How can you contribute to the fight against tick-borne diseases in your community? Consider participating in citizen science initiatives, educating others about tick prevention, and advocating for policies that support vector-borne disease research and control efforts.

Together, we can overcome the obstacles in tick pathogen research and work towards a future where the burden of these diseases is significantly reduced.