Adam Birkenheuer

Primary focus of this project is the cure and prevention of cytauxzoonosis.

Primary focus of this project is to discover and evaluate Cytauxzoon felis genes and proteins for the purpose of diagnostic assays, vaccine antigens and novel drug targets.

Cytauxzoon felis is an important tick-borne protozoal parasite that causes fatal disease in domestic cats. Acute cytauxzoonosis has a mortality rate of 40-50% even with the best medical management. Tick control is the only preventive measure for the infection, but it is expensive and pet owner compliance is notoriously poor. Vaccination is an attractive alternative, as it is more cost-effective and has potential for a long duration of immunity. However, no vaccine for C. felis exists. Unlike many other pathogens, C. felis has never been successfully cultured in vitro, which severely limits our ability to study the parasite without experimentally infecting cats. Infecting cats to study C. felis raises major ethical concerns as the infected cats develop severe illness that can lead to death or euthanasia. Our long-term goals are to improve our ability to prevent, treat, and diagnose cytauxzoonosis. Therefore, it is critical for us establish alternative methods to cultivate C. felis as a foundation to achieve these goals. In contrast to previous failed attempts which focused on the feline-associated life stages, we are developing a novel approach utilizing ticks and tick cell lines as the basis to cultivate C. felis. Similar techniques have been used for other closely related protozoan parasites, like Babesia and Plasmodium. Laboratory-based systems to propagate C. felis will serve as a source for future vaccine development as well as a platform to screen for more effective treatments for cytauxzoonosis. We hypothesize that in vivo and in vitro cultivation of C. felis can be established in ticks and tick cell lines. In Specific Aim 1, we will infect ticks with C. felis via direct injection. In Specific Aim 2, we will establish in vitro cultivation of C. felis in tick cell lines. Experimental Design and Methods: Specific Aim 1: We will infect A. americanum (the primary vector host for C. felis) by directly injecting C. felis-infected feline erythrocytes via 2 routes: 1) Direct injection of C. felis into the tick hemocoele, or 2) Direct injection into the digestive tract through the anal pore to more closely mimic the natural life cycle. Sample sizes have been determined (300 ticks per injection method) to detect which route of infection has the highest infection rate and at which point in time each method reaches its maximal infection rate. Whole tick histology sections and saliva will be serially evaluated for infection weekly up to 6 weeks post injection. Saline-injected ticks will serve as negative controls for these experiments. Specific Aim 2: We will be culturing C. felis in tick cell lines and assessing 3 different variables. These variables include 2 different A. americanum cell lines, 2 different temperatures (simulating tick attachment to the host and post-detachment conditions) and the effect of gametogenesis (sexual reproduction of the parasite within the tick) on the growth of C. felis in tick cells. The growth of C. felis in both aims will be monitored and verified via direct visualization (light and electron microscopy), in situ hybridization (ISH), and polymerase chain reaction.

Boehringer-Ingelheim Vetmedica (hereafter referred to as ����������������BIVI���������������) is providing funding for a Postdoctoral Scholar in the Department of Clinical Sciences College of Veterinary Medicine at North Carolina State University (hereafter referred to as the ����������������Department���������������). Services for the scholar include: 1. Writing technical bulletins 2. Presenting evening lectures externally to veterinarians and technicians and internally to students, interns, and residents 3. Providing expert opinion advice to veterinarians, technicians and pet owners through telephone and email consults 4. Assisting the professional services veterinarians, marketing veterinarians and VeTS group through telephone or email consultation on adverse event cases 5. Assisting with media events that require expert opinion 6. Attend the annual ACVIM meeting at which time resident scholars and the manager of this BIVI program will hold an annual meeting.

Funds are for post-doc (resident position-Jessica Pritchard) to which 20% of time will be to provide the following services: 1. Writing technical bulletins 2. Presenting evening lectures externally to veterinarians and technicians and internally to students, interns, and residents 3. Providing expert opinion advice to veterinarians, technicians and pet owners through telephone and email consults 4. Assisting the professional services veterinarians, marketing veterinarians and PeTS group through telephone or email consultation on adverse event cases 5. Assisting with media events that require expert opinion 6. Attend the annual ACVIM meeting at which time resident scholars and the manager of this BIVI program will hold an annual meeting.

The mortality rate in immune-mediated hemolytic anemia (IMHA) in the dog is reported to be as high as 70%, and over 50% of these deaths are attributed to thromboembolism. Thus, a readily accessible and accurate method to monitor anticoagulant therapy is urgently needed to improve survival. In a recent on-line survey of 27 United States veterinary colleges performed by the PI, 95% of the 19 respondents reported their institution used unfractionated heparin (UFH), with or without antiplatelet therapy, for thromboprophylaxis in hospitalized dogs with IMHA. Most (70%) survey respondents monitored UFH therapy using activated partial thromboplastin times (aPTT). Although aPTT is easy to use, variations in reagents and instrumentation create significant disparities in its measured response. Measurement of the amidolytic activity of the plasma protease Factor Xa, which is blocked by UFH, is considered the gold standard for heparin monitoring. Yet, our survey indicates that few veterinary colleges (3/19) had the ability to perform this assay and only 1 reports its routine use. The assay is complex, with inherent background activity, and is not cost effective without running a large number of samples, making its use unlikely to become widespread or available cageside. The survey also revealed that most (79%) respondents had access to in-house thromboelastography (TEG). Our long-term goal is to utilize a whole blood viscoelastic-based assay, such as TEG, to provide objective point-of-care measurements of clot kinetics and strength to more effectively titrate thromboprophylaxis with UFH therapy. An increased understanding of the importance of TEG monitoring for managing UFH therapy in IMHA patients is expected to result in an immediate improvement in survival. This cost effective diagnostic aide is likely to gain widespread use in the veterinary referral setting, fostering the dissemination of information to general practitioners

Cytauxzoonosis is a rapidly emerging hemoprotozoan disease with an expanding geographic range. Transmitted by ticks, the initial phase of parasite replication is responsible for clinical illness. During this stage of parasitemia, most infected cat develops an acute illness best characterized as protozoal sepsis. Disseminated intravascular coagulation, acute respiratory distress syndrome, multiple organ dysfunction, and death often follow. Recently, treatment involving supportive care plus the combination of the antimalarial drug atovaquone with the antibiotic azithromycin was demonstrated to result in a 60% survival of naturally infected cats. This compares well to a 26% survival for cats treated with imidocarb dipropionate, or 5% survival in cats provided supportive care alone. Unfortunately, atovaquone is not widely available, expensive, and difficult to administer. Further, there are strains of C. felis that are resistant to the drug. Recently, the authors have treated a small number of both experimentally and naturally infected cats with an alternative antimalarial drug, Coartem���������, and 7/9 of the treated cats survived the illness. Coartem has the advantage of being far less expensive and easier to administer than atovaquone, requiring only the administration of one tablet twice daily for three days. If Coartem is not inferior to atovaquone and azithromycin for the treatment of cytauxzoonosis, it would offer a substantial advantage in terms of cost and ease of administration, thereby facilitating the successful treatment of more cats. This prospective, randomized open-label trial will compare the efficacy of atovaquone and azithromycin with Coartem for the treatment of cats with naturally occurring cytauxzoonosis.

The first objective of this project is to evaluate novel treatments of Cytauxzoon felis infections in domestic cats. To this end a randomized-controlled trial will be performed treating naturally occurring cytauxzoonosis in client-owned cats. The second objective of this project is to establish in vitro cultures of Cytauxzoon felis for genetic analyses, diagnostic test development and vaccine development.

Cytauxzoonosis is a life-threatening disease of domestic cats caused by the tick-transmitted apicomplexan protozoan parasite Cytauxzoon felis that is closely related to Theileria, Babesia and Plasmodium (the causative agent of malaria). Without treatment 97% cats with cytauxzoonosis die. Even with the best available treatments, morbidity is extreme and mortality rates approach 40%. Regardless of outcome, treatment can cost thousands of dollars per case. Since its discovery in the 1970s, the geographic distribution of this parasite has grown rapidly and cytauxzoonosis is now diagnosed in 35% of the states in the continental USA. The distribution of C. felis is likely to expand further and threaten even more cats as the primary vector, the lone star tick, extends its distribution. The high mortality and growing epidemic point to vaccination as the only practical control strategy. Unfortunately no vaccine against C. felis exists. In fact, prior to our work, no C. felis antigens had ever been characterized. Nonetheless, evidence does suggest that cats surviving infection develop protective immunity. Two major barriers to antigen discovery and subsequent vaccine development include the inability to culture C. felis in vitro and a lack of funding for feline specific diseases. Federal agencies focused on human and food animal health stopped funding C. felis research once it was recognized in the early 1980s that cytauxzoonosis was limited to domestic and wild felids. In order to overcome the limitations posed by an inability to culture the parasite in vitro, we have recently sequenced, assembled and annotated the entire 9.1 Mbp C. felis genome. This has enabled us to identify ¡Ö 4,300 C. felis protein-coding genes, each of which represents a possible protective antigen. Our challenge now is to identify and prioritize which of these genes are the best vaccine candidates. We hypothesize that high throughput immunoscreening using C. felis protein microarrays will result in the rapid identification and prioritization of a large number of potentially protective C. felis antigens representing excellent targets for a C. felis vaccine.

While working in Dr. Adam Birkenheuer?s lab, I hope to continue work in further investigating the pathogenesis of tick-borne protozoan parasites, including Cytauxzoon felis. C. felis is highly virulent parasite which infects domestic and wild felids in the Southeastern United States. Without treatment, this disease is nearly 100% fatal in domestic cats, as cats decline rapidly from the disease after only 1 week of clinical symptoms. Even with the best treatments, which cost thousands of dollars, the mortality rate from this infection is still 40%. This highly pathogenic disease is important to study from a number of standpoints, including improving feline health as well as from a comparative medicine perspective. Cytauxzoon is closely related to a number of pathogens that infect a wide range of both animals and people, including Plasmodium falciparum, the causative agent of malaria. Any new information uncovered about Cytauxzoon could have implications for diseases that impact humans, livestock, companion animals, and wildlife.