Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 2

A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for a Masters degree. Onderstepoort, 1994 / Anaplasmosis is a tick transmitted haemoparasitic disease of bovines, caused by A. marginale and A. centrale, which occurs worldwide and results in losses of significant economic importance. Bovines that survive infection are immune to reinfection with a homologous isolate and partially protected against heterologous isolates. South African isolates of A.marginale and A. centrale are compared. [Abbreviated Abstract. Open document to view full version] / MT2017

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 37-44)

Digitized by Kansas State University Libraries

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Johann F. Coetzee / The Office International des Epizooties (OIE) Animal Health Code categorizes bovine anaplasmosis as a notifiable disease. Many species of the genus Anaplasma cause anaplasmosis. Co-infections with two or more Anaplasma spp. occur in cattle. A competitive ELISA is regarded as a reliable test for identifying A. marginale-infected cattle. However, cross-reactivity among related Anaplasma spp. has been reported when using cELISA. In the absence of effective treatment strategies and vaccine availability, anaplasmosis control strategies are primarily focused on disease identification and prevention and development of chemosterilization strategies. Four studies were completed to improve the diagnosis, treatment, and control of bovine anaplasmosis. In the first study, a real-time qRT-PCR was developed to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. This detection limit was equitable to the minimum infective unit of one A. marginale bacterium. In the second study, qRT-PCR results determined needle-free injection was superior to needle injection for controlling iatrogenic transmission of A. marginale in cattle. The qRT-PCR demonstrated 100% sensitivity by 21 days post-infection and 21 days prior to 100% sensitivity with cELISA. The third study determined the pharmacokinetic parameters of chlortetracycline in group fed, ruminating Holstein steers: volume of distribution (40.9 L⁄kg); rate constant (0.0478 h-1); dose-normalized area under the curve (0.29 h•µg⁄L); clearance (1.8 L⁄kg⁄h); elimination half-life (16.2 h); maximum concentration/dose (4.5 ng⁄mL); and time of maximum concentration (23.3 h). Dose linearity was confirmed for oral chlortetracycline dosages of 4.4, 11, and 22 mg/kg/day. The final study established an in vivo pharmacokinetic-pharmacodynamic relationship between chlortetracycline and anaplasmosis carrier clearance in bovine plasma (85.3 ng/mL). The qRT-PCR confirmed chemosterilization of all oral chlortetracycline-treated cattle within 49 days of treatment. Furthermore, qRT-PCR was an effective alternative to the subinoculation of splenectomized cattle for accurate and precise disease classification. The diagnosis, treatment, and control of anaplasmosis were enhanced through the application of qRT-PCR. Further studies are necessary for determining the mechanism of action between chlortetracycline binding to the 30S ribosome of A. marginale and carrier clearance.

The American dog tick, Dermacentor variabilis, is an obligate blood feeding ectoparasite. This tick is a known vector of pathogens that affect the health of wildlife, humans, and livestock and is abundant in Manitoba. The etiological agent of bovine anaplasmosis, Anaplasma marginale, along with members of the spotted fever group rickettsiae are bacteria that are transmitted by this tick. I examined the distribution of these bacteria in Manitoba’s tick population using molecular techniques. During the eradication of an outbreak of bovine anaplasmosis in Manitoba, there was no evidence the bacterium had spilled over into the tick population. Rickettsia montanensis was detected with a mean prevalence of infection of 9.8% (range, 0.00 - 21.74% among localities) in 8 of 10 localities within the province. It was also determined that 19.9% (SE ±1.14) of adult questing ticks collected in one vector season overwintered through to the next spring.