A set of studies was designed in order to better understand the exposure of horses in Canada to Ixodes-borne diseases, namely equine granulocytic anaplasmosis (EGA, caused by Anaplasma phagocytophilum) and Lyme borreliosis (LB, caused by Borrelia burgdorferi). In the first study, equine serum samples submitted to veterinary diagnostic laboratories in SK, MB and ON were tested for antibodies against A. phagocytophilum and B. burgdorferi, using the point-of-care SNAP® 4Dx® ELISA. Horses seropositive to EGA were found in SK and MB and horses seropositive to LB were found in SK, MB and ON. Overall seroprevalence according to the SNAP® 4Dx® ELISA was 0.53% for EGA and 1.6% for LB. Samples that tested positive for antibodies against A. phagocytophilum (n=2) and B. burgdorferi (n=6) by SNAP® 4Dx® ELISA and 2 randomly selected subsets of samples that tested negative (n=92 each) were then re-tested using currently recommended serologic methods, and test results were compared. A lack of agreement was found between the SNAP® 4Dx® ELISA and indirect immunofluorescent assay (IFA) for EGA (McNemar test p = 0.000001). Agreement of the SNAP® 4Dx® ELISA and ELISA confirmed with Western Blot (WB) for LB was only fair (Kappa 0.23). Due to the lack of agreement between serologic tests for EGA and LB in the first study, another study to further evaluate the agreement among available serologic tests was conducted. A set of 50 convenience serum samples submitted to the veterinary diagnostic laboratory in SK was tested by SNAP® 4Dx® Plus ELISA for antibodies against A. phagocytophilum and B. burgdorferi. Samples were also tested by IFA for antibodies against A. phagocytophilum in two referral laboratories, and by IFA, ELISA confirmed with WB and Equine Lyme multiplex assay for antibodies against B. burgdorferi in three referral laboratories. Again, test results varied between the different tests. For EGA, all 3 pair-wise test comparisons lacked agreement. For LB, agreement between tests ranged from poor to fair. Differences in test methodology and antigens used, cut-off settings between the laboratories and false positive or false negative results are likely the cause for the different assessment of the same sample as seropositive or seronegative. In the third study, the goal was to describe potential risk factors for exposure of horses in Canada to EGA and LB. Management factors in horses that tested seropositive or seronegative for EGA or LB, respectively, in the previous studies were evaluated. Horse owners were surveyed with regard to their horses’ signalment, timing of pasture housing, and province of residence, travel history, tick infestation history, history of Lyme vaccination and history of previously diagnosed tick-borne disease. Response rate (11.5%) and the number of seropositive horses available for evaluation were low, which precluded statistical analysis. The majority of seropositive horses resided in SK, was pastured in the fall, did not have a recent travel history and had not had visible tick infestation. These observations supported exposure of horses to tick-borne diseases within Canada. Potential risk factors require further investigation. As information about tick infestation in horses is scarce in general, a passive surveillance study of horse ticks in SK was conducted in 2012 and 2013. A total of 833 ticks from over 86 horses were received. All ticks were Dermacentor species, i.e. D. albipictus, D. andersoni and D. variabilis. D. albipictus ticks were mostly received in February and March, D. andersoni mainly in April and June and D. variabilis mostly in May and June. Geographic distribution of the species in SK was similar to that previously reported based on active and passive surveillance. No Ixodes species were received.

Background: Equine granulocytic anaplasmosis (EGA), caused by the organism Anaplasma phagocytophilum, is a tick-borne disease of clinical importance in Southwest Virginia. The disease is recognized worldwide and causes pyrexia, anorexia, limb edema, and lethargy. Diagnosis in endemic areas is often based on clinical signs, but confirmation of infection can be made via detection of morulae on a peripheral blood smear or polymerase chain reaction analysis (PCR) at the time of disease or by serologic detection of antibodies 2-4 weeks post infection. There is growing interest in stall-side methods for diagnosis of various equine diseases which has led to an increased use of the SNAP 4DX Plus Test® for vector-borne diseases. Objectives: Determine seroprevalence of antibodies to A. phagocytophilum in the equine population of Southwest Virginia and changes in seroprevalence compared to samples taken 6 years earlier. Determine the percentage of horses with clinical signs consistent with EGA that were positive for A. phagocytophilum infection and assess common presenting clinical signs, hematologic variables, and confirmatory diagnostic test results. Animals: Seroprevalence was evaluated in horses presented for routine annual Coggins testing in 2013 and 2019-2020. Clinical features of disease and diagnostic test results were evaluated in horses presenting with clinical signs compatible with A. phagocytophilum infection from September 2019-August 2020. Methods: Seroprevalence was determined using the IDEXX SNAP 4DX Plus Test® on serum collected from horses presenting for annual Coggins testing in 2013 and 2019-2020. Samples collected in 2013 had been stored at -7580 degrees F since collection. Age, sex, county of residence, and month of sampling were statistically analyzed in the seroprevalence population. Horses presenting with clinical disease consistent with EGA from September 2019-August 2020 had the following diagnostic tests performed: complete blood count (CBC), blood smear for morulae detection, polymerase chain reaction (PCR) analysis, immunofluorescence antibody testing (IFAT), and the IDEXX SNAP 4DX Plus Test®. Results: Seroprevalence of A. phagocytophilum in the equine population of Southwest Virginia increased from 8.5% in 2013 to 11.2% in 2019-2020, although this increase was not statistically significant. In the 2019-2020 population, month of sampling was significantly associated with presence of antibodies to A. phagocytophilum. Positive samples were more common from November-February than other times of the year. When the two sample time periods were combined, sex was significantly associated with presence of antibodies to A. phagocytophilum with geldings more likely to be seropositive. Within the clinical case population, 35% of horses with clinical signs compatible with equine granulocytic anaplasmosis had confirmed infection. The most common hematologic abnormality in affected horses was thrombocytopenia. PCR analysis was the most sensitive diagnostic test to diagnose infection followed by identification of morulae on blood smears. Conclusions: Seroprevalence of A. phagocytophilum is similar to other endemic areas in the United States and appears to be increasing over time. In active clinical cases, diagnosis is best made via PCR or detection of morulae on a blood smear. The SNAP 4DX Plus Test® was not appropriate for diagnosis of active EGA in acute cases. Seroprevalence of Anaplasma phagocytophilum in the equine population of Southwest Virginia / Master of Science / Equine granulocytic anaplasmosis (EGA) is a common tick-borne disease in the United States and worldwide. The causative bacteria, Anaplasma phagocytophilum, also infects humans, dogs, and various domestic animal species. In horses, signs of disease include fever, decreased appetite, leg swelling, and depression. Diagnostic testing that is both accurate and timely is still lacking. The point-of-care SNAP 4DX Plus Test® used to diagnose vector-borne infectious disease in dogs has been suggested for similar use in horses. The objectives of the current study were to determine seroprevalence of antibodies to A. phagocytophilum in the equine population of Southwest Virginia and to characterize the clinical signs and diagnostic test findings of horse with clinical signs of EGA. Seroprevalence was determined using the SNAP 4DX Plus Test®. Serum samples were obtained from horses presenting for annual Coggins testing in 2019-2020. Samples from 2013 were also tested to determine if seroprevalence had increased. Horses presenting with clinical signs consistent with A. phagocytophilum were examined by a veterinarian and had blood drawn for a complete blood count (CBC), blood smear evaluation, polymerase chain reaction analysis (PCR), immunofluorescent antibody testing (IFAT), and the SNAP 4DX Plus Test®. Seroprevalence in 2019-2020 was 11.2% and 8.5% in 2013. This is similar to other endemic areas in the United States and Europe. In horses sampled from 2019-2020, the month of sampling was significantly associated with presence of antibodies to A. phagocytophilum with most of the positive samples being identified in November through February. Geldings were more likely to be seropositive than mares. Thirty five percent of horses with signs consistent with EGA were confirmed to have the disease. Within this population, PCR analysis and/or detection of morulae on the blood smear were reliable indicators of disease while diagnostic techniques utilizing serology were unreliable. This is the first study to determine seroprevalence of A. phagocytophilum in Southwest Virginia. In the actively infected population, PCR and blood smear evaluation remain the most sensitive methods of diagnosis. While the SNAP 4DX Plus Test® is useful for serologic data collection, it was not appropriate for acute diagnosis of EGA.

Lyme borreliosis was first described some 30 years ago in the USA. Today, it is the most common vector borne disease in Europe and the USA. The disease can have multiple stages and symptoms can manifest from various parts of the body; joints, skin heart and nervous system. In Europe, neuroborreliosis is the most frequent late stage diagnosis. Although Lyme borreliosis is treatable with antibiotics and the causative spirochete has not been shown to be resistant to drugs, some patients do not recover completely. They have persistent symptoms and are diagnosed with chronic or persistent Lyme borreliosis. The mechanism behind the lingering symptoms is unclear but might be due to tissue damage caused by the immune system. The aim of this thesis was to study the immunological differences between patients with different outcome of Lyme borreliosis, i.e. chronic, subacute and asymptomatic, and various factors that might influence the course of the disease. The Borrelia-specific IFN-γ and IL-4 secretion was detected in blood and cerebrospinal fluid from patients with chronic and subacute neuroborreliosis during the course of the disease. Blood samples were also obtained from patients with erythema migrans (EM) and acrodermatitis chronicum atrophicans. An early increase of IFN-γ with a later switch to an IL-4 response was observed in patients with a subacute disease course whereas the IFN-γ secretion continued to be elevated in chronic patients. The Borrelia-specific Th1-response was further investigated in chronic, subacute and asymptomatic individuals by studying the expression of the Th1-marker IL-12Rβ2, on a protein and mRNA level. The cytokine secretion and Foxp3, a marker for regulatory T-cells, were also analyzed. Chronic patients had a lower IL-12Rβ2 expression on CD8+ T-cells and a lower number of Borrelia-specific IFN-γ secreting cells compared to asymptomatic individuals. Chronic patients also displayed a higher expression of Borrelia-specific Foxp3 than healthy controls. The conclusions for these tow studies were that a strong Th1-response early in the infection with a later switch to a Th2-response is beneficiary whereas a slow or weak Th1-response corresponds to a prolonged disease course. The influence of a previous infection with another pathogen, seen to suppress the immune response in animals, and the possible gender difference in immune response was also investigated. Patients with EM were screened for antibodies to Anaplasma phagocytophilum (Ap) as a sign of a previous exposure to these tick-borne bacteria. Blood lymphocytes from Ap seronegative, Ap seropositive and healthy controls were stimulated with Borrelia antigen and the secretion of IL-4, IL-5, IL-12, IL-13 and IFN-γ was detected by ELISPOT. Ap seropositive patients had a lower number of cells responding with IL-12 secretion compared to the other groups which might indicate an inhibited Th1-response. Reinfections with Lyme borreliosis was in a previous study, done by Bennet et al, found to be more frequent in postmenopausal women than in men. To investigate if there was an immunological explanation to the gender discrepancy, blood lymphocytes from individuals reinfected with Lyme borreliosis and individuals infected only once were stimulated with various antigens. The cytokine secretion was detected by ELISPOT, ELISA and Immulite. There were no differences between reinfected and single infected individuals. However, women, regardless of times infected, displayed a Th2-derived and anti-inflammatory spontaneous immune response compared to men. A previous infection with the bacteria Ap might possibly have a long term effect on the immune system and might be of disadvantage when mounting a Th1-response to a Borrelia infection. Also, the Th2-derived response displayed by postmenopausal women could indicate why more women than men get reinfected with Borrelia burgdorferi. / On the day of the public defence date of the doctoral thesis the status of article III was Accepted; the status of article IV was Submitted and the title was "Importance of induction and secretion of interferon-gamma for optimal resolution of human Lyme borreliosis: differencesbetween different outcomes of the infection".