Team:TU Eindhoven/Policy Practices/Application scenarios





Application scenarios



Using aptamers as recognition elements for our COMBs enables the COMBs to sense virtually all biomarkers for which aptamers have been generated. These targets include toxins, whole cells, viruses and proteins. To give an overview of the huge impact our membrane sensors might have on society, we sketch three scenarios where our COMBs may be applied. For these scenarios, we have reached out to many stakeholders to gain a clear image of what the problems are that society currently faces and how our membrane sensors can play a role in solving these problems.

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Intestinal use


The bacteria which make up our life-sustaining microbiota in our guts secrete small molecules to convince the immune system not to attack them. This communication can be seen as one-way-traffic, but the possibility of making it bilateral is interesting, to say the least. See how our device can assist in enabling us to communicate with our gut bacteria.

Q Fever


From 2007-2009, the largest Q Fever outbreak ever recorded took place in The Netherlands. Over 4.000 people became infected with the Q Fever causing pathogen Coxiella Burnetii. Q Fever still remains very hard to diagnose, making prevention easier to accomplish than cure. See how our device can help in detecting Q Fever.

Countering pesticide overuse

Agricultural developments enabled civilization to expand to the industrial society we know today. These developments, however, have not always been harmless. The overuse of pesticides as a prevention to protect our crops is an example of a harmful practice. In an ideal world, pesticides would only be present where pathogens are present. See how our device can be a first step towards this ideal world.



Countering pesticide overuse



Recent agricultural developments have enabled human civilization to expand to the industrial society as we know it today. As we are dealing with a growing population, the use of products to enlarge agricultural production is crucial. Root diseases such as rot lead to great crop losses and have been responsible for great famine in the past [1] [2]. Plants do not fully grow and the fruit production significantly diminishes [1]. It is estimated that about 30-50% of food is lost, due to inadequate agriculture. Root diseases, mostly caused by fungal growth, can sometimes lead to 100% loss of crops. Fungi, such as Fusarium, and fungal-like eukaryotic microorganisms called oomocytes, such as Pythium and Phytophthora, modulate plant physiology and defense mechanisms via the secretion of virulence factors and effectors [2]. [3].. One of the main invasive approaches is the secretion of Cell Wall Degrading Enzymes (CWDE). These proteins disrupt the cell wall structure of plant cells, thereby leaving the cells vulnerable for infiltration by pathogenic organisms [4]. Several different methods for pathogen detection have been developed. Some of them make use of specific fungal antigens as diagnostic markers. Next to that, the production of mycotoxins and allergens by these pathogens could possibly also be used for detection [3].

However, developments to increase agricultural yield are not always harmless. A particular example of a dangerous agricultural development is the overuse of pesticides.
Pesticides is a broad term to describe any agent (chemical or biological) that is capable of disturbing pathogens in their normal functioning. These pathogens can be microorganisms, herbs, insects or fungi, each of them forming a threat to the growth of crops. Since fungi form the most prominent threat to roots, we will take a closer look at the functionality of pesticides specific for fungi: fungicides. Chemical fungicides make use of chemical molecules to intervene in pathways crucial for fungal growth or preservation. Chemical fungicides have several different modes of action. They can act on pathways involved in chitin and sterol biosynthesis, microtubule assembly and mitochondrial respiration [2][9]. Also research is done towards a new method making use of antimicrobial peptides. These peptides disrupt membrane structure by binding to and disordering the double layer cell membrane of fungi, bacteria or viruses. Some of them are capable of activating intracellular pathways by entering cells or binding to specific receptors [9]. Because the cell wall structure of oomycetes is different from that of fungi, most fungicides are not effective against oomycetes. To fight oomycete infection phenylamide metalaxyl, inhibiting RNA Polymerase 1 is used. More research is done to find different molecular routes that can be used as center of attack [2].
Pesticides are effective in inhibiting pathogen growth, but the extensive usage also forms one of the main environmental issues in the world today. Pesticides can pollute drinking water [5]. Wild plants and animals become endangered with threat for extinction [6]. Besides this environmental damage, there is evidence that pesticides are hormone disruptive and possibly even lead to cancer [7][8]. Finally there are also cost issues, which were emphasized by regulators during the symposium at the RIVM.





Current approach



Pesticides are used to protect crops against harmful microorganisms. The chemicals can either be spread preventively or afterwards as cure [2]. Preventive usage of chemicals could imply unnecessary contamination of the soil. Whereas in curative usage, it is necessary to keep constant check of the soil and crop quality. This, if done manually, is very labor intensive, expensive and in any cases crop quality diminishes due to infection. In an ideal world, pesticides distribution only occurs when pathogens are present. Thereby it would be useful if soil check-up and pesticide distribution is automated.





Application COMBs



To reduce pesticide levels in the soil we could use our sensor system to monitor for presence of pathogens. Using the “click reaction” to adhere pathogen-specific aptamers to our sensor, we could manage the detection of pests in crops. This detection can then activate the process in the cell, which eventually leads to the excretion of pesticides. In this way pesticides will only be secreted when this is necessary, which can reduce the environmental burden. To make our system work, we need a molecule that we can detect and we need to be able to produce a useful output signal. As discussed before, pathogenic organisms produce virulent factors such as mycotoxins. The factors produced by these pathogens are specific, can diffuse through the ground and are small enough to be detected using our aptamer system [10]. As output-signal our bacteria should produce pesticides. Therefore we could make use of a TEF-protease at the intracellular domain. The cleaving off of a transcription factor could trigger the transcription of a specific gene encoding for a pesticide. Subsequent translation of the mRNA produced will form the necessary pesticides. Especially antimicrobial peptides could easily be produced making use of bacterial mechanisms. By adding the appropriate signals to the pesticide, it will be transported out of the bacteria cell and into the surroundings. There it will attack the pathogen and protect the plant roots.

Of course one should consider the risks when bringing a genetically modified organism into the environment. To learn more about safety measures we consulted with Frank van der Wilk from COGEM. He stimulated us to think about a safe way to bring our bacteria into the environment. A possible solution could be to put our pesticide producing bacteria in beads that can be spread across the land inside fertilizer for example.[see safety beads
For the safety part of our project we produced alginate beads as designed by the iGEM team of Paris Bettencourt (2012). Therefore, polymer formation is used to encapsulate the potentially harmful bacteria. The advantage of using this alginate bead is that it captures the bacteria, but still allows for the diffusion of possibly useful molecules from the surroundings. A disadvantage, that could form a problem for our application in pesticide secretion, is that the material has a low mechanical strength and chemical durability. Polymer chemistry is one of the most quickly developing fields and there are multiple ways to tune polymer properties. Mechanical strength can for example be tuned by the branching rate, where an increase in branching leads to a higher stress-strain ratio. Modulations to the polymer backbone can in turn increase chemical durability, by reducing oxidative susceptibility [11]. A variety of different porous materials is developed for different purposes [12].
] However, this would entail the risk of the beads being eaten by animals and might lead to contamination of different lands. An alternative solution could be to produce a tube of a similar material that can be spread across the land. This could avoid contact between our bacteria and animals. Also it could create an easy method for renewing bacteria, by simply putting fluid containing our bacteria in the hose at one end. In either case it is important that the material used is semipermeable. This means it would allow for (products of) pathogens to diffuse into the tube and for pesticides to diffuse out of the tube, but it would be impossible for the bacteria to escape. The material should be wear resistant. If using beads, it should be able to pass through the gastrointestinal tract of an animal and come out unharmed. But also it should not degrade due to weather circumstances or because of microorganisms or compounds in the soil.





[1] Penn State College of Agricultural Sciences, “Fungal Root Rots And Chemical Fungicide Use”, Penn State Extension, pp. 1-4, 2015.
[2] Attard, A., Gourgues M., Galiana E., Panabières F., Ponchet M. and Keller H., “Strategies of attack and defense in plant–oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan)”, Journal of Plant Physiology, vol. 165, no. 1, pp. 83-94, Sept. 2007.
[3] Thornton C.R. and Wills O.E., “Immunodetection of fungal and oomycete pathogens: Established and emerging threats to human health, animal welfare and global food security”, Critical Reviews in Microbiology, vol. 41, no. 1, pp. 27-51, Jun. 2013.
[4] Blackman L.M., Cullerne D.P. and Hardham A.R., “Bioinformatic characterisation of genes encoding cell wall degrading enzymes in the Phytophthora parasitica genome”, BMC Genomics, vol. 15, no. 1, pp. 1-24, Sept. 2014.
[5] Power A.G., “Ecosystem services and agriculture: tradeoffs and synergies”, Philosophical Transaction of the Royal Society, vol. 365, no. 1554, pp 2959–71, Sept 2010.
[6] Berny P., “Pesticides and the intoxication of wild animals”, Journal of Veterinary Pharmacology, vol. 30, no. 2, pp. 93-100, Apr. 2007.
[7] Khalil N., Chen A. and Lee M., “Endocrine disruptive compounds and cardio-metabolic risk factors in children”, Current Opinion in Pharmacology, vol. 19, no. 1, pp. 120-124, Oct. 2014.
[8] L’Héritier F., Marques M., Fauteux M. and Gaudreau L., “Defining Molecular Sensors to Assess Long-Term Effects of Pesticides on Carcinogenesis”, International Journal of Molecular Sciences, vol. 15, no. 9, pp. 17148-61, Sept. 2014.
[9] Hayes B.M., Anderson M.A., Traven A., Van der Weerden N.L. and Bleackley M.R., “Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins”, Cellular and Molecular Life Sciences, vol. 71, no. 14, pp. 2651-66, Jul. 2014.
[10] Turner N.W., Subrahmanyamb S. and Piletsky S.A., “Analytical methods for determination of mycotoxins: a review”, Analytica Chimica Acta, vol. 632, no. 2, pp 168-180, Jan. 2009.
[11] Young R.J. and Lovell P.A., “Introduction to Polymers”, CRC Press, 3th edition, Jun. 2011.
[12] Nischang I., “Porous polymer monoliths: Morphology, porous properties, polymer nanoscale gel structure and their impact on chromatographic performance”, Journal of Chromatography A, vol. 1287, no. 1, pp. 39-58, Apr. 2013.



Intestinal use



The gastrointestinal tract is packed with up to a 100 trillion microbes, called the microbiota [1]. This life-sustaining microbiota assists in producing essential vitamins and the digestion of important nutrients. Perhaps the most promising discovery is that these microbes play an essen¬tial role in protection against pathogens and communication with our immune system [2]. Disturbances within the communication of the microbiota and our immune system, is associated with many different pathologies, such as Crohn’s disease, colon cancer and even obesity. A specific, reliable and rapid sensor for these diseases and biomarkers is still lacking. The development of our device can thereby be a first step towards such a diagnostic tool, and in the distant future even for treatment of these pathologies.
We have talked to the ICC (Initiative on Crohn and Colitis) and gastroenterologists from the Academic Medical Center (AMC) in Amsterdam about the feasibility of our sensor and the aspects in our system that can be a point to concern. Also Joost Drenth, head of the department Stomach-Colon and Liver diseases at the UMC St. Radboud in Nijmegen, and Alexandra Ginsberg, designer and writer in the field of synthetic biology, gave us their opinion and advice about our project.





Current approach



Intestinal cancer


Intestinal cancer, also called colorectal cancer or colon cancer is the third most common cancer diagnosed in both men and woman in the U.S. [3]. The disease usually starts with the growth of polyps
Polyps are small clumps of cells formed in the lining of the colon. They are different variants and can turn into the formation of tumors. Benign polyps are not fatal and often termed as hyperplastic polyps. These polyps remain benign and the chance that these will turn into cancer is very low. Adenomatous and hamartomatous polyps are non-malignant variants; however, they have a chance of becoming cancerous if not removed. The last variant of polyps is an invasive variant and can turn into tumors. When polyps evolve into cancer, the uncontrolled tissue growth of the tumor can be seen through all tissue layers of the colon rectum. Even the spreading of cancer to other organs is possible in this stage.
in the mucosa, which is the innermost lining of the intestines. The mucosa consists of epithelium cells, loose connective tissue, and forms the first line of defense in the intestines.
Currently, intestinal cancer is diagnosed and screened by performing fecal occult blood tests (FOBT), a sigmoidoscopy or colonoscopy. A FOBT is the first screening method in which the feces are being checked for the presence of small amounts of blood that are not immediately visible. Even though FOBT is feasible, accepted and widely available, its sensitivity for polyps is low and it also has a relatively low specificity. This led to many false-positive results and repetitive screening is often necessary [4]. A FOBT screening is often combined with a sigmoidoscopy. This method of screening uses a lighted tube with a camera to examine the rectum and sigmoid colon. With a small biopsy tool, samples of tissue can be collected for further examination. Patients need to empty their bowel completely for the screening and therefore patients cannot eat solid food for several days beforehand to ensure accurate results. This manner of screening is invasive for patients and thus not preferred if it is not necessary [5]. Thirdly, a colonoscopy can be executed to detect and immediately remove precancerous polyps. A thin, flexible tube with a camera is used to examine the colon (large intestines) and the lower part of the small intestines. The only difference with a sigmoidoscopy and the benefit of a colonoscopy is that it can inspect the whole colon instead of only a small part [6]. Even though the colonoscopy is currently the most accurate and used screening method, patients often need to be sedated and before the test they need to empty the colon which means no solid food and taking laxatives for several days.

Inflammatory diseases: Crohn’s disease and Colitis


In both Crohn’s disease and Colitis, inflammations occur along the gastro-intestinal tract. The causes for these diseases are still unclear; however, it seems that it is triggered by an oversensitive or damaged immune system. Therefore these diseases belong to the immune mediated diseases, which means the body isn’t attacking its own substances but it’s triggered by external factors. For both diseases, first a physical examination is performed to see whether patients have the corresponding symptoms, such as blood in the stool. Afterwards, lab tests are performed to check for signs of inflammations and infections, for example to check white blood cells or blood protein levels. Then the decisive method is a colonoscopy and CT scans from the abdomen [7][8]. The disadvantage of diagnosing these inflammatory diseases is that there is no one single technique that gives a correct diagnosis, but the combination of multiple methods is needed for an accurate result.





Future application: COMBs



Seen the problems with the current diagnostic tools, an accurate, non-invasive and early detecting sensor is needed. This can be realized by finding the right biomarkers. Currently the medical world is in search for a detecting mechanism that can sense human hemoglobin in the intestines. Blood’s main component is hemoglobin; therefore it is a crucial biomarker for various abnormalities in the intestines, such as colon cancer, Crohn’s disease, etc. However, human hemoglobin is a biomarker for diverse irregularities in the intestines, whereby still follow-up research has to be done after positive results. From gastroenterologists of the AMC Amsterdam we got the suggestion of COX-2, an enzyme that can be found in the intestines at an early stage of colon cancer. Both Joost Drenth and experts from the ICC saw opportunities of Calprotectin as another biomarker, which is a protein that is associated with inflammatory diseases, such as Crohn’s disease and Colitis. Calprotectin is specifically correspond to inflammatory diseases and thus can distinguish inflammatory diseases from irritable bowel syndrome, which has the same symptoms. By designing aptamers that specifically bind to human hemoglobin, COX-2 or calprotectin, the biosensor can be implemented in the intestines. It can sense the actual situation in the intestines and detect the biomarkers accurately and at an early stage instead of identifying it afterwards in the feces. The high specificity of aptamers is crucial, because other variants, such as bovine hemoglobin, can be found in the intestines as well. As mentioned above, the distinction between different intestinal diseases can be made directly within one test.
Our biosensor can be combined with auto fluorescence imaging. This imaging technique (combined with endoscopy) uses auto fluorescence to detect polyps whereby the intestinal tract lights up as green and the thickenings in the colon become purple. This current technique is not reliable as it doesn’t detect the polyps. By using fluorescent proteins on the intracellular side of our biosensor and the right biomarker, our sensor can accurately detect polyps and secrete a fluorescent signal that can be observed with this imaging technique. Furthermore, in a more distant future, the biosensor can release visible colors when it has detected a biomarker that for example can easily be observed in the feces. This would be easily for people to do at home and facilitates the large scale screening within a population. Besides detection, a more promising development is the secretion of medicine using COMBs when it has detected a biomarker. The detection of biomarkers can lead to the release of transcription factors which induces various signaling pathways in the bacteria causing the biosensor to produce medicine. There are some critical notions on this: the bacteria must be contained and cannot colonize within the intestines. This can be accomplished by bringing the bacteria into alginate beads. As described on our safety page.
Additionally, medicines have strict criteria, whereby each package needs to be equal and have the same dose. This is difficult to realize when working with living organisms. An option is to store the medicine in compartments in the bacteria. Another problem which has to be taken into account in future research is the presence of DNAses and RNAses in the intestines. These proteases are able to go through the pores of the alginate beads and will break down the aptamers.
Even though there are still many obstacles, it is believed to be the prospective manner of taking and secreting medicine. Therefore, permissions to do research with GMOs in the intestines are given more often these days. For example, the COGEM has given permission to perform an I-phase trial on a study about the secretion of IL-10 by GMOs in the intestines, to target treatment of Crohn’s disease. The first tests on humans are already done with promising results [9]. Another example is the study of programmable bacteria, which can detect and record an environmental signal in the mammalian gut [10]. These studies offer good prospects for our COMBs in the future.





[1] Ray K., “Gut microbiota: Married to our gut microbiota”, Nat. Rev. Gastroenterol. Hepatol., vol. 9, no. 10, pp. 555, Oct. 2012.
[2] Mazmanian S.K., Round J.L. and Kasper D.L., “A microbial symbiosis factor prevents intestinal inflammatory disease”, Nature, vol. 453, no. 7195, pp. 620–5, May 2008.
[3] Cancer of the Colon and Rectum - SEER Stat Fact Sheets. (n.d.). Retrieved September 14, 2015, from http://seer.cancer.gov/statfacts/html/colorect.html
[4] Bond J. H., "Fecal occult blood test screening for colorectal cancer", Gastrointestinal Endoscopy Clinics of North America, vol. 12, no. 1, pp. 11–21. Jan. 2002.
[5] Sigmoidoscopy: MedlinePlus Medical Encyclopedia. (n.d.). Retrieved September 13, 2015, from https://www.nlm.nih.gov/medlineplus/ency/article/003885.htm
[6] Colonoscopy. (n.d.). Retrieved September 13, 2015, from http://www.webmd.com/colorectal-cancer/colonoscopy-16695
[7] Diagnosing Crohn’s Disease: Lab Tests, Imaging Tests, X-Rays, and More. (n.d.). Retrieved September 14, 2015, from http://www.webmd.com/ibd-crohns-disease/crohns-disease/crohns-disease-diagnosis
[8] Colitis Symptoms, Causes, Treatment - How is colitis diagnosed? - MedicineNet. (n.d.). Retrieved September 14, 2015, from http://www.medicinenet.com/colitis/page7.htm
[9] Braat H. et al., "A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease", Clinical Gastroenterology and Hepatology, vol. 4, no. 6, pp. 754-9, Jun. 2006.
[10] Kotula J. W. et al., "Programmable bacteria detect and record an environmental signal in the mammalian gut", Proceedings of the National Academy of Sciences, vol. 111, no. 13, pp. 4838-43, Feb. 2014.



Q Fever



Q fever is a zoonotic disease which is caused by Coxiella burnetii, a pathogen which usually resides in cattle and ticks and can be transmitted to humans through aerosols and raw animal products. The high susceptibility of humans for the pathogen was recognized by both the Soviet Union and the United States, who devised to use the organism as a biological weapon [1]. The high infectivity also resulted in numerous transient Q fever outbreaks in animals and humans within the European Union. Concerns were raised, however, in the European Union as the Netherlands was hit with the largest outbreak ever recorded with 4,026 human cases notified between 2007 and 2010 [2].


The most important source of Q fever appears to be goats and sheep. The primary symptom of affected goats and sheep is abortion and reduced fertility. This abortion results in the release of over a billion C. burnetii bacteria per gram of placenta, providing a major risk for public health. Such abortions were first observed in the Netherlands in 2005 at two dairy goat herds. From 2005 till 2007, fifteen Q fever abortions were diagnosed. Q fever had, however, spread through the Netherlands more transiently, as antibody screening showed that C. burnetii antibodies were detected on 57% of the 344 farms. [3].

From 2007 onward, the first human cases of Q fever were reported in the province of Noord-Brabant, taking place after visits to dairy goat farms where spontaneous abortions had occurred. The epidemic became apparent after general practitioners reported on the occurrence of pneumonia within patients not responding to standard antibiotics. The outbreak in 2007 marked the beginning of a course of other outbreaks of Q fever throughout the province of Noord-Brabant (Figure 1). At the moment, still eight of these companies remain infected [4].

Figure 1: Between 2007 and 2010, the Netherlands was hit with the largest outbreak of Q fever ever recorded with 4,026 human cases notified. Most of these cases were reported in Noord-Brabant, the Dutch province in which our university is located. This image was adapted from RIVM.

We have talked to Dr. Roest, a veterinary microbiologist who is strongly involved in research to Q fever and Anja Garritsen, a scientist working at InnatOss, one of the biggest research institutes in the field of Q fever in the world, to get a better insight into the important aspects around Q fever. Besides that we spoke to Jeanette van der Ven, owner of a goat’s farm near Eindhoven and one of the board members of the ZLTO (association for entrepreneurs in the green sector), to get inspiration and feedback from people who were really involved in this problem. Additionally we spoke to René van den Brom, a researcher working for the national institute that focusses on animal well-being, who did his thesis about Q fever. He told us about the problems he faced in his research and showed us his results.





Current Approach



Transmission


The extreme infectivity of Coxiella burnetii stems from its ability to shift from a Small Cell Variant to a Large Cell Variant. In the former state, C. burnetii is metabolically inactive and extremely resistant within the environment. When the Small Cell Variant enters a cell through phagocytosis, it will shift to its active large cell form which is metabolically active (Figure 2). This metabolically active form will replicate in the Coxiella Containing Vacuole (CCV). Eventually, the metabolically active large variants will undergo sporogenic differentiation to produce the resistant Small Cell Variants. Upon cell lysis, these are released into the environment where they can survive for longer periods and infect other organisms [5]. C. burnetii enters the cell through phagocytosis. The bacterium is internalized in the nascent Coxiella Containing Vacuole (CCV), which rapidly merges with auto phagosomes and lysosomes. Normally, the acidification of the phagosome results in degradation of bacteria. C. burnetii, however, is very resistant to numerous harsh environmental conditions. Acidification therefore does not degrade the bacterium: it even triggers the bacterium to become metabolically active. As a result, the CCV can grow out to a mature vacuole. This image was adapted from Nature Reviews

Transmission of the pathogen to mammals can occur in a number of ways. The most important route of transmission to humans is formed by contaminated aerosols. Important sources of these aerosols are goat feces, contaminated wool and a placenta shed by an infected animal [6]. Due to C. burnetii’s high infectivity, inhalation of a single infected particle can be enough to be infected by the pathogen (Figure 3).

Figure 3: The most important source of Q fever infection during the Q fever outbreak in the Netherlands was goat manure. Goats are a perfect reservoir for C. burnetii and if they are infected, their manure contains the small cell variant of the pathogen. In windy conditions, aerosols of the manure can form which can infect humans once breathed in.


Pathology


The main challenge of diagnosis of Q fever is its atypicality. Contact with C. burnetii is followed by a 2-3 week incubation period. After the incubation period symptoms may occur, even though most cases of Q fever remain asymptomatic (60%) [7]. Even if the patient shows symptoms, these symptoms are often very atypical. The infection is characterized by the unpredictability and diversity of its symptoms. Examples of symptoms which may or may not occur within a single patient are fever, headaches, hepatitis and meningoencephalitis. This clinical polymorphism impedes an early and accurate detection of Q fever. This is very problematic, as failure to diagnose Q fever early and accurately can lead to the manifestation of chronic Q fever (see Figure 4). Figure 4: After contact with Coxiella burnetii, the pathogen enters the cell. After an incubation period of 2-3 weeks, 40% of the patients begin to show symptoms. These symptoms manifest in a wide range of forms, but pneumonia is by far the most common. The other 60% of the patients do not show acute symptoms. If the infection goes untreated, it may become chronic. This can lead to multiple complications, including abortion and endocarditis.

Diagnostics


Currently, diagnostic tools available for Q fever are aimed at a few biomarkers. The first biomarker is the DNA of C. burnetii. By amplifying a part of the genome of C. burnetii within the patient’s blood trough PCR acute Q fever infection can be detected [8]. The test is highly specific and very sensitive, but only as long as DNA remains detectable within the patient’s blood. The DNA can approximately be detected until two weeks after contact with the pathogen within blood samples of the patient. After these two weeks, the patient has typically become immune against the pathogen and the DNA of the pathogen can no longer be found within the patient’s blood. This is problematic as Q fever’s clinical polymorphism impedes doctors to rapidly recognize Q fever. Therefore, prevention of Q fever infection is often easier to accomplish than cure. The second biomarker is a direct result of this built up immunity. Two types of antibodies become detectable in the blood. In contrast to the DNA of C. burnetii, these antibodies remain detectable within the blood for longer periods (Figure 5) [9].A drawback of serological tests is the lag phase in antibody response: it takes up to two weeks before a patient’s blood gives detectable levels of Phase I-antibodies. Therefore, these tests can only be used to detect previous Q fever rather than acute Q fever. Additionally, serological tests and skin tests suffer from a higher variability and show long return times. Besides that, the success of skin tests relies on trained personnel. As an alternative to these tests, interferon gamma detection has been developed [10]. In this test, a blood sample from the patient is taken which is incubated with C. burnetii antigens. Rather than testing for the presence of antibodies, however, the immunity of the patient is tested by interferon gamma detection, a signaling molecule which is secreted by immune cells upon recognition of the pathogen. The developed test shows similar specificity and sensitivity as skin tests and serological tests. Data is, however, available within 24 hours and the test does not depend on training of personnel. A drawback in the diagnostics of Q fever is that the distinction between patients with an acute infection or patients which already have been through the disease process is hard to make. For microbiologists this is a very important thing, because they are only allowed to give antibiotics to patients with acute Q fever. At a later stage only treatment in psychological aspect can be done. Figure 5: After first contact with the pathogen, 40% of the patients fall ill. After the patient falls ill, DNA of Coxiella Burnetti becomes detectable in the blood. This DNA remains detectable for approximately 2 weeks. 1.5 weeks after the acute infection, Phase I-antibodies become detectable in the blood. In an acute infection, this is rapidly followed by detectable levels of Phase II-antibodies. In contrast to Coxiella Burnetii’s DNA, serological tests for antibodies remain positive for more than 12 months after the acute infection.





Future application: COMBs



Goat vaccination


The biomarkers available for Q fever have led to a wide range of diagnostical tools. Conventional tools to detect previous Q fever have been serological tests and skin tests. Both tests, however, have major drawbacks. Serological tests suffer from a higher variability and show long return times. Skin tests also suffer from a higher variability and success of skin tests relies on trained personnel [Specific Interferon gamma Detection]. As an alternative to these tests, interferon gamma detection has been developed. In this test, a blood sample from the patient is taken which is incubated with Coxiella Burnetti antigens. Rather than testing for the presence of antibodies, however, the immunity of the patient is tested by interferon gamma detection, a signaling molecule which is secreted by immune cells upon recognition of the pathogen. The developed test shows similar specificity and sensitivity as skin tests and serological tests. Data is, however, available within 24 hours and the test does not depend on training of personnel.

Both serology and the interferon gamma detection test have a major disadvantage, however, as they rely on immunology of the patient. This is a problem because of the lag phase in antibody response: it takes up to two weeks before a patient’s blood gives detectable levels of Phase I-antibodies. Therefore, these tests can only be used to detect previous Q fever rather than acute Q fever. An alternative test which can be used to detect acute Q fever infection is real-time PCR. During this test, part of Coxiella Burnetti’s genome within the patient’s blood is amplified through PCR. The test is highly specific and very sensitive, but only as long as DNA remains detectable within the patient’s blood. This is a disadvantage since DNA is only detectable for approximately two weeks after the acute infection.

Numerous serological tests have been developed to target Q fever. Whereas a wide range of tests has become available to detect previous Q fever accurately, these tests can only be used to detect previous Q fever. Therefore, these tests are very important to map Q fever epidemics by assessing whether a person should be vaccinated or not. These tests, however, fail to address acute infections of Q fever.
A test which can detect acute infections of Q fever, the real-time PCR test, can provide an early and accurate diagnosis of Q fever. The problem with this test, however, is that it relies on the presence of Coxiella Burnetii’s DNA in the blood of the patient. Since this DNA can only be detected for about two weeks after the patient falls ill, such a real-time PCR test should be conducted shortly after a patient falls ill. This is problematic as Q fever’s clinical polymorphism impedes doctors to rapidly recognize Q fever. Therefore, prevention of Q fever infection is often easier to accomplish than cure.

In 2009 at the peak of the Q fever outbreak, the Dutch government decided to offer risk groups who lived near animal farms vaccines against Q fever.[11]In addition to offering vaccines to risk groups, the Dutch government mandated the culling of pregnant goats at farms which were known to be infected, as pregnant goats presented a major threat to public health. All in all, tens thousands of pregnant goats were killed to stop the Q fever outbreak, even though it was not tested whether the pregnant goats were infected. The decreasing numbers of human cases of Q fever in 2010 might indicate that the measures taken by the Dutch government have been effective at limiting the Q fever epidemic [12].
In addition to culling of ten thousands of pregnant goats in 2009, the Dutch government imposed numerous regulations on goat farms to limit the outbreak. These measures include a mandatory vaccination of dairy sheep and dairy goats, mandatory tank milk monitoring for Q fever every 2 weeks and a ban on removing manure from the farm. Many of these regulations are still enforced: dairy goats and sheep have to be vaccinated yearly, there is a report duty for signs of Q fever and every dairy farm is obligated to take part in a biweekly bulk tank milk test for the presence of C. burnetii as the milk of infected goats contains DNA from the pathogen. Figure 6: The milk of goats infected with C. burnetii contains its DNA. In addition to the DNA of the pathogen, the milk usually contains the Phase I- and Phase II-antibodies which are active against C. burnetii. The tank milk test, however, consists of a real-time PCR test as the antibodies are present because of the vaccination of goats.
Jeanette van der Ven, owner of a goat’s farm near Eindhoven and one of the board members of the ZLTO (association for entrepreneurs in the green sector), told us that it is pretty hard for farmers to gain monsters for the testing of their goats. Protocols have to be followed and everything has to be disinfected, which is not pleasant for the animals. It would be a great improvement to make the milk tests easier to perform. With the use of COMBs the farmers will be able to test the milk at home by bringing our sensor system, encapsulated in alginate beads, into the milk samples. These samples can be simply extracted from the milk tanks or even be built into the milk tanks. When antibodies are present, the beads shall turn into a color. This way, farmers don’t have to send milk samples to a research institute anymore and will get the result of the test immediately. Treatments can be started immediately and there will be no more risk of newly infected cattle in the meantime. Because all goats are vaccinated, detection of antibodies into the milk does not always signify infection. However, experts from the InnatOss told us that they vaccinate once a year, but often notice that previous vaccines are not working anymore. With the use of COMBs, real-time detection of antibodies is possible, which is really helpful by determine when the goats have to be vaccinated again. Also in this case the farmers are able to do these tests themselves. We hope that with the use of COMBs a main problem for the society can be solved, by detecting infections easily at an early stage and optimize the vaccination program.





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