AKC/CHF Tick Grant Updates

Last year, the American Shih Tzu Club Charitable Trust donated $5,000 to to the AKC Canine Health Foundation’s tick-borne project, and were pleased that AKC matched our donation
Below are reports from current active grants that received funding from that AKC/CHF tick-borne project. Thank you to all donors to our Charitable Trust who make such donations to canine health projects possible.

ASTC Charitable Trust Directors: Dan Haley, Carlene Snyder, Patricia Thomas, and Sally Vilas
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Grant 02284-A: Lyme Disease in Dogs: Prevalence, Clinical Illness, and Prognosis
Principal Investigator: Jason Stull, VMD, PhD Research Institution: Ohio State University | Grant Amount: $14,148.00

Start Date: 7/1/2016 | End Date: 6/30/2018 | Progress Report: Mid-Year 1
Report Due: 12/31/2016 | Report Received: 1/13/2017

Original Project Description:
Lyme disease (or Borreliosis) is a bacterial disease of dogs and humans that is transmitted by tick bites. In people, Lyme is the most common tick-transmitted disease in the US, with over 25,000 cases in 2014. While most common in the northeastern coastal states and the upper Midwest, Lyme disease is moving into other regions of the U.S. and Canada. Dogs infected with Lyme disease rarely show signs of illness (typically lameness), but can be severe (e.g., kidney disease). Diagnosis, treatment and prevention of Lyme disease in dogs are complicated by limited research and conflicting professional guidance. Current practices may unnecessarily place dogs at risk for illness and negative outcomes.

The investigators will follow a large group of dogs from different regions of the U.S. and Canada. During this period the investigators will determine how often healthy dogs test positive for Lyme disease (meaning they have been bitten by an infected tick) and identify how often they later develop a Lyme-related illness. The risks and benefits of management strategies for Lyme-positive dogs and obstacles to effective tick prevention will be determined to help clarify unmet pet owner education needs. These findings are likely to extend to better understanding of canine and human Lyme disease, and improve health outcomes. Collectively, this work will allow us to identify, define and improve upon best practices for prevention and control of Lyme disease in areas with different Lyme risks, ultimately improving the health of dogs and people.

Publications:
None to date

Report to Grant Sponsor from Investigator:
Consistent with the timeline provided in our proposal, to-date we have developed and begun pre- testing a dog owner questionnaire. The PI has had conversations with veterinarians at several clinics in Maine and Ohio. Several clinics have tentatively agreed to participate in the study beginning Spring 2017. The human subject’s protocol has been started and estimated to be submitted within the next 2 weeks. This project is on track to begin enrolling dogs and owners this spring and to accomplish our stated objectives within the 2-year period.
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Grant 02285-A: Thrombocytopenia and Occult Vector-Borne Disease in Greyhound Dogs: Implications for Clinical Cases and Blood Donors
Principal Investigator: Linda Kidd, DVM, PhD | Research Institution: Western University of Health Sciences | Grant Amount: $12,960.00

Start Date: 7/1/2016 | End Date: 6/30/2017 | Progress Report: FINAL
Report Due: 6/30/2017 | Report Received: 6/30/2017

(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Retired racing Greyhounds (RRG) are popular pets, and also commonly serve as blood donors to treat all types of dogs. Not all Greyhounds are RRG; show-bred Greyhounds (SBG) have traceable pedigrees verifying they do not descend from racing lines. Low platelet (thrombocytopenia) and white blood cell counts are considered normal findings in Greyhounds. Protein in the urine is common. Because these findings can also be caused by infection with vector-borne disease agents, Greyhounds can present clinicians with a diagnostic dilemma. Whether these laboratory results are found with the same frequency in RRG and SBG has not been investigated.

Racing Greyhounds are commonly exposed to the brown dog tick, which transmits many agents that can cause disease. Vector-borne diseases are also transmitted by the lone star tick, also common in the region of the U.S. where racing farms are located. Because these pathogens can cause chronic, clinically silent infection, the investigators hypothesize that infection occurs in, and contributes to blood and urine abnormalities in some healthy-appearing RRG. This study will compare the prevalence of vector-borne diseases in RRG and SBG, determine whether thrombocytopenia, low white blood cell counts and protein in the urine are associated with vector-borne disease in RRG, and whether blood and urine abnormalities occur with the same frequency in RRG and SBG. The results will help veterinarians decide when to pursue infectious disease testing, and whether more aggressive infectious disease screening for both pet and blood donor Greyhounds is warranted based on lineage.

Publications:
Kidd, L., Qurollo, B., Lappin, M., Richter, K., Hart, J. R., Hill, S., … Breitschwerdt, E. B. (2017). Prevalence of Vector-Borne Pathogens in Southern California Dogs With Clinical and Laboratory Abnormalities Consistent With Immune-Mediated Disease. Journal of Veterinary Internal Medicine, 31(4), 1081–1090. https://doi.org/10.1111/jvim.14735

Report to Grant Sponsor from Investigator: Retired racing greyhounds are popular pets commonly used as blood donors. However, not all greyhounds are RRG. Show-bred greyhounds have traceable pedigrees verifying they do not descend from racing lines. Low platelet and white blood cell counts are considered normal findings in greyhounds. Protein in the urine is also common in greyhounds. Because these findings are also caused by infection with vector borne disease agents, greyhounds can present clinicians with a diagnostic dilemma. Whether these abnormalities are found with the same frequency in RRG and SBG has not been investigated. Racing greyhounds are commonly exposed to the brown dog tick (Rhipicephalus sanguineus). This tick transmits many agents that cause disease. Additional kinds of vector borne disease are transmitted by the lone star tick, also common in the region of the US where racing farms are located. Because these organisms can cause chronic, clinically silent infection, we hypothesized that infection occurs in, and contributes to blood and urine abnormalities in some healthy appearing retired racing greyhounds.

The objectives of this study were to compare the prevalence of vector borne disease in retired racing greyhounds and show bred greyhounds using highly sensitive tests; to determine whether low platelets, low white blood cell counts and protein in the urine are associated with vector borne disease in retired racing greyhounds, and if these blood and urine abnormalities occur with the same frequency in retired racing greyhounds and show bred greyhounds.

We enrolled 30 retired racing greyhounds and 28 show bred greyhounds in the study. We found that healthy appearing retired racing greyhounds were exposed to or infected with vector borne disease agents significantly more than show bred greyhounds. This information will help clinicians because it shows they can use racing history to help guide the scope of diagnostic testing for vector-borne disease in greyhounds with compatible clinical signs or in blood donor screening.

Like other studies, we found that Babesia vogeli (formerly Babesia canis) and Ehrlichia canis exposure in the retired racing greyhounds. In addition to transmitting Babesia vogeli and Ehrlichia canis, evidence suggests some Bartonella species and Mycoplasma species may be transmitted by Rh. sanguineus. This is the first study testing for exposure to and infection with Bartonella and Mycoplasma species in greyhound dogs. Three retired racing greyhounds tested seropositive to Bartonella species, and three retired racing greyhounds tested PCR positive to M. haemocanis. As we have found in a previous AKC funded study, (Kidd et al 2017) and another study (Maggi et al 2014) these results illustrate the importance of comprehensive screening for vector borne disease. Further studies of the impact of chronic infection with these agents on the health of greyhounds and other breeds is warranted.

We have also analyzed the data regarding the relationship of platelet count, low white blood cell counts and urine protein levels to greyhound lineage and exposure to vector borne disease and will allow it to undergo peer review before publishing the results. Manuscript preparation should be complete August 2017. We plan on submitting to the Journal of Veterinary Internal Medicine.
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Grant 02295-A: The Role of Lymphocytes in Canine Monocytic Ehrlichiosis
Principal Investigator: Mary Anna Anna Thrall, DVM, MS | Research Institution: Ross University School of Veterinary Medicine | Grant Amount: $15,000.00

Start Date: 7/1/2016 | End Date: 12/31/2017 | Progress Report: End-Year 1
Report Due: 6/30/2017 | Report Received: 6/30/2017

(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Canine monocytic ehrlichiosis (CME) is a serious disease of dogs, caused by the intracellular bacteria Ehrlichia canis that is transmitted by a tick bite. There is no vaccine for CME, and the pathophysiology of why the disease is more serious in some dogs is not understood. CME is very common in St. Kitts, home to Ross University School of Veterinary Medicine. The large numbers of affected dogs are a valuable resource for studies of this important disease. Lymphocytes (a type of white blood cell) appear to be related to the pathophysiology of CME. The investigators will study the types of lymphocytes present in dogs with both mild and severe disease and compare them to non-affected dogs. Lymphocytes will be identified by type as B or T cells using antibody markers for lymphocytes and flow cytometry.

The investigators will determine if an increase in lymphocyte counts (lymphocytosis) is associated with severity of disease, and whether clonality (having a large number of the exact same type of lymphocyte) is associated with severity of disease. Fifty Ehrlichia-positive dogs admitted to Ross University will be evaluated for their number of lymphocytes by blood cell counts, by flow cytometry to determine their lymphocyte subsets, and by PCR and antibody testing for the presence of tick-borne disease. These dogs will be compared to healthy control dogs. The researchers will also evaluate 50 dogs presenting with persistent lymphocytosis and determine the percentage of those dogs that are Ehrlichia positive. The findings of this study will advance understanding of the pathophysiology and diagnosis of ehrlichiosis and lymphocytosis.

Publications:
None to date

Report to Grant Sponsor from Investigator:
Canine monocytic ehrlichiosis (CME) is a serious disease of dogs, caused by the intracellular bacteria Ehrlichia canis that is transmitted by a tick bite. Lymphocytes (a type of white blood cell) appear to be related to the pathophysiology of CME. We are studying the types of lymphocytes present in dogs with both mild and severe disease and comparing them to non-affected dogs. Lymphocytes will be identified by type as B or T cells by flow cytometry. T cells are an important defense against intracellular pathogens; they typically kill pathogens by secreting cytokines and releasing cytotoxic granules. However, the Ehrlichia organism often successfully survives in untreated dogs. This may be because they are actually living within the T cells. Also, T cells can contribute to an excessive immune response and cause immune mediated damage, which is likely the cause of the increased globulin concentrations and destruction of platelets that we see very commonly in Ehrlichia-positive dogs. B lymphocytes are responsible for secreting antibodies and presenting antigen, and they also secrete cytokines.

We have not completed the study due to an equipment failure (flow cytometry), but we are now progressing rapidly, using an instrument at Colorado State University. We have evaluated 35 dogs and have determined that 10 of the 35 dogs have increased lymphocyte concentrations. Eighteen of the 35 dogs have increased concentrations of B cells, and 16 have increased concentration of T cells. Three dogs have expansions of one type of lymphocyte that is similar to what is seen with lymphocytic leukemia. Thirteen have increased concentrations of both. Thirty-two of the 35 dogs have decreased platelets, cells important for clotting, likely due to immune-mediated destruction, and 17 are anemic. Seventeen dogs are hyperproteinemic. (See Table 1) We will now evaluate 15 additional dogs, and by re-testing all 50 dogs after antibiotic therapy, we will determine if therapy has resulted in returning the laboratory results to normal. We will also now determine if the dogs that have lymphocyte abnormalities are more severely affected. In the future, we will examine the separated T cells to see if they are harboring Ehrlichia organisms. We have requested a 6 month no cost extension to complete the study. The findings of this study will advance understanding of the pathophysiology and diagnosis of ehrlichiosis and lymphocytosis.
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Grant 02287: Enhanced Testing for the Diagnosis of Bartonellosis in Dogs
Principal Investigator: Edward B Breitschwerdt, DVM Research Institution: North Carolina State University | Grant Amount: $103,013.00

Start Date: 8/1/2016 | End Date: 7/31/2018 | Progress Report: End-Year 1
Report Due: 7/31/2017 | Report Received: 7/31/2017

(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

Bartonellosis, a zoonotic bacterial disease of worldwide distribution, is caused by approximately 10 different Bartonella species. Bartonella are transmitted to canines and humans by ticks, fleas, lice, mites, and sand flies. Dr. Breitschwerdt’s laboratory demonstrated the first evidence for Bartonella infections in dogs in 1993. Bartonella species have been associated with an expanding spectrum of important disease manifestations including anemia, endocarditis, hepatitis, lymphadenitis, myocarditis, thrombocytopenia and vascular tumor-like lesions. Infections can be life-threatening. Due to a lack of sensitive and reliable diagnostic assays, definitive diagnosis of bartonellosis in dogs remains a significant problem. Because these bacteria invade cells and infect tissues throughout the body, this chronic intracellular infection is difficult to cure with currently used antibiotic regimens. This study will develop improved serodiagnostic tests for bartonellosis in dogs. These assays can also be used for world-wide sero-epidemiological prevalence studies, and to establish early and accurate diagnosis. Dr. Breitschwerdt’s research group has described concurrent infection in dogs, their owners and veterinary workers; this allows for a One Health approach to this important emerging infectious disease.

Publications:
None to date

Report to Grant Sponsor from Investigator:
This AKC-CHF funded research has resulted in substantial refinement of our Bartonella serodiagnostic testing knowledge of healthy dogs (pets, working dogs and potential blood donors) and sick dogs being evaluated for a differential diagnosis of Bartonellosis. Based upon testing and comparing results using eight different Bartonella species or strains, we were able to idenfify two species that when used in combination should increase serodiagnostic sensitivity compared to the three strains that we have used diagnostically for the past decade. As these are not the two species currently in use diagnostically, our historical testing most likely underestimated the prevalence of Bartonella antibodies in seroepidemiological studies published by our research group and more importantly failed to report positive Bartonella spp. serological results that could be used to guide therapy in a sick dog. In both our research and diagnostic laboratory testing, we have always adhered to the adage: The kindest form of therapy is an accurate diagnosis. This study also allowed us to begin to assess the utility of another serodiagnostic technique called Western immunblotting for assessment of a dog’s exposure to a Bartonella spp. WB interpretation can be challenging and this study has allowed us to compare IFA and WB sensitivities and to define minimal critera for reporting a “positive” WB. Finally, our efforts to date have not identify specific combinations of small Bartonella proteins (peptides) that have diagnostic utility as an inhouse rapid assay that could be used by veterinarians to rapidly determine if a dog is exposed to a Bartonella spp. We are currently extending our efforts to assess additional peptide combinations in an effort to improve assay sensitivity. This brief paragraph does not do justice to the numerous hours of research effort that was made possible because of the research funding support from the American Kennel Club Canine Health Foundation or the dogs and their owners who will ultimately benefit from the findings that were made.
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Grant 02292: Broad-Range Detection of Canine Tick-Borne Disease and Improved Diagnostics Using Next-Generation Sequencing
Principal Investigator: Pedro Paul Diniz, DVM, PhD | Research Institution: Western University of Health Sciences | Grant Amount: $60,717.00

Start Date: 9/1/2016 | End Date: 2/28/2018 | Progress Report: End-Year 1
Report Due: 8/31/2017 | Report Received: 8/30/2017

(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Diagnostic tests based on the detection of DNA of infectious organisms from clinical samples have revolutionized veterinary medicine in the last decades. Currently, diagnostic panels for several tick- borne organisms are available through universities and private laboratories in the USA and abroad. However, the vast majority of results from clinically ill dogs are negative for tick-borne diseases, which frustrates veterinarians and dog owners trying to reach a definitive diagnosis and improve treatment options. These panels are based on the detection of previously known DNA sequences of each pathogen, with little room for detecting new organisms. Consequently, the current assays may suffer from "myopia": a self-fulfilling effect that prevents the detection of new or emerging organisms. Using an innovative approach, the investigators will employ next-generation sequencing (NGS) to overcome the limitations of current diagnostic technology. With NGS, the investigators can generate millions of individual gene sequencing reads from each clinical sample, allowing for the identification and characterization of multiple organisms from a single sample. Testing samples from dogs naturally exposed to tick-borne diseases, NGS will detect not only new organisms but also characterize genetic differences among known organisms. The resulting dataset of a large number of DNA sequences of known tick-borne organisms and previously undetected organisms in naturally-infected dogs will support the development of diagnostic tools to simultaneously advance canine and human health.

Publications:
Manuscript under preparation:
Persico E., Qurollo B., Thomas B., Hegarty B., Breitschwerdt E., Diniz P.P.V.P. Molecular prevalence of selected canine vector-borne pathogens in the United States (2008-2015). Journal of American Veterinary Medical Association.
Vasconcelos E., Oakley B., Diniz, P.P.V.P. Strategies for assessing vector-borne diseases 16S rRNA next generation sequencing data in veterinary clinical samples.

BMC Microbiology or BMC Veterinary Research.
Vasconcelos E., Oakley, B., Billeter SA, Jett LA, Wournell AL, Kjemtrup AM, Padgett KA, Yoshimizu MH, Metzger ME, Barr MC, Diniz PPVP. Epidemiology of flea-borne microbiomes detected in northern and southern California by universal 16S rRNA generation sequencing. Veterinary Microbiology.

Report to Grant Sponsor from Investigator:
Dogs from any breed, age or gender can be infected with microbes transmitted by ticks or fleas. These diseases can cause devastating effects and even death not only to dogs but also to humans. Ticks are present everywhere in the US, and they bring the risk of transmitting the microbes to dogs and humans. In fact, dogs infected with these microbes have been reported at every US state including Hawaii, and more recently, Alaska. It is still very difficult to diagnose these conditions, and approximately 95% of suspected dog cases are negative when we use current diagnostic technology. One of the biggest limiting factors for the development of better diagnostic tools is the insufficient funding opportunities for large-scale projects. CHF has implemented a new funding mechanism to address such limitation, which supports this study. The long-term goal of our research team is to expand the current diagnostic tools to include a larger spectrum of potentially hazardous microorganisms. Our innovative approach is based on four pillars: (1) large-scale DNA sequencing to identify known and potentially new organisms present in blood of 500 dogs naturally exposed to vector-borne diseases; (2) increase in sensitivity and specificity of large-scale sequencing by targeting major families of potentially hazardous organisms, (3) advanced bioinformatic analysis of billions of DNA sequences from a large number of dogs suspected of infection; and (4) comprehensive quality- control measures in order to support and validate the impact of our results. In the last six months, expanded the sample size to 537, to account for missing samples. We have completed the preparation of the samples from all canine samples, and have submitted them for deep DNA sequencing. We also design novel family-level primers for the Anaplasmataceae, Bartonellaceae and Mycoplasmataceae families using a unique computational approach and confirmed their sensitivity and specificity based on rigorous benchtop experiments. We sought further training to update and expand our computational protocols to remain at the cutting-edge workflow of analysis of large-scale DNA sequences in metagenomics. Our bioinformatics pipeline is ready to quickly analyze the raw data coming from our large-scale DNA sequencing effort. Finally, we already identified key elements to improve the use of this new technology in veterinary diagnostics. Our results will probably become a paradigm shift in the detection and identification of the cause of vector-borne diseases in dogs, by unveiling thousands of novel potentially hazardous microorganisms. Our data will serve as the new foundation for the implementation and expansion of novel diagnostic methods, and will ultimately support early diagnosis and better medical care to dogs worldwide. By sponsoring this CHF research initiatives, breed clubs and other sponsors will have a positive impact in animal health at a global level.
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Grant 01780: Defining the Mechanism by Which Ticks Locate Dogs in Order to Better Prevent Disease Transmission Principal Investigator: Emma Weeks, PhD Research Institution: University of Florida | Grant Amount: $104,867.31
Start Date: 3/1/2013 | End Date: 2/28/2018 | Progress Report: End-Year 4
Report Due: 2/28/2017 | Report Received: 1/13/2017

(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
The brown dog tick (BDT) is common across the U.S. and is the most widely distributed tick in the world. BDT's carry and transmit the pathogens that cause debilitating diseases such as canine ehrlichiosis and babesiosis. Prevention of these diseases is accomplished through tick control. BDT's can complete their entire life cycle indoors, making management difficult. Records of infestations are increasing and unpublished data indicates that a high level of pesticide resistance is present in domestic populations. Consequently once introduced, these ticks are particularly hard to eradicate and as one female tick may lay 5,000 eggs, the problem soon gets out-of-hand. Pesticide resistance leads to aggressive treatment regimes, which in turn, lead to increased exposure of humans and pets to chemical residues. Alternatives to pesticides are needed. Studies have shown that BDT's are attracted to dog odor, a blend of volatile chemicals used by ticks to find a blood meal. In this study, Dr. Weeks will identify the chemicals BDT's use to locate a dog. This will enable manipulation of tick behavior thereby facilitating management and reducing the need for extensive use of pesticides. Improved tick control without the need for increased environmental pesticide applications will improve the quality of life for dogs and their owners.

Publications:
None at this time.

Report to Grant Sponsor from Investigator:
The brown dog tick (BDT) is common across the US and the most widely distributed tick in the world. BDT’s are capable vectors of pathogens that cause canine ehrlichiosis and babesiosis as well as other disease agents. Prevention of these diseases is accomplished through tick control. BDT’s can complete their entire life cycle indoors, making management difficult. Records of infestations are increasing and unpublished data indicates that a high level of acaricide resistance is present in domestic populations.

Consequently once introduced, these ticks are particularly hard to eradicate and as one female tick may lay 5,000 eggs, the problem soon gets out-of-hand. Acaricide resistance leads to aggressive treatment regimes, which in turn, leads to increased exposure of humans and pets to acaricide residues. Alternatives to pesticide applications are needed. Studies have shown that BDT’s are attracted to dog odor, a blend of volatile chemicals used by ticks to find a blood meal. Identification of the chemicals BDT’s use to locate a dog (semiochemicals) would enable manipulation of tick behavior thereby facilitating management and reducing the need for extensive use of acaricides. Improved tick control without the need for increased acaricide applications will improve the quality of life for dogs and their owners or handlers. Work will be accomplished through four successive objectives to 1) collect dog odor, 2) identify chemicals that ticks can detect, 3) test chemicals for tick attraction and ultimately 4) evaluate efficacy of an attractant-based tick trap.

For the first objective, the collection of dog odor, all animals have been identified and the samples have been collected and analyzed by chromatographic techniques. Furthermore the chemicals have been identified tentatively by mass spectrometry. For the second objectives the electrophysiological techniques have been established and ten ticks have been tested against each dog breed sample plus a mixed sample of all dog breeds (Total 60 ticks). For the third objectives the behavioral assay has been established. Attraction has been demonstrated to whole dog hair samples and positive controls. Further studies will continue to test the response of ticks using behavioral and electrophysiological means to determine the biologically-active chemicals in dog odor. These chemicals will then be tested in the behavioral bioassay and in traps.

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