Novel vaccine strategies for bovine anaplasmosis

Abstract

Bovine anaplasmosis is a mild to severe hemolytic disease caused by the intraerythrocytic pathogen Anaplasma marginale (Am). Transmission of bacteria occurs biologically via ticks and mechanically via blood-contaminated fomites and biting flies. Following inoculation, cattle hosts exhibit persistent bacteremia and can serve as reservoirs for subsequent infection. Bovine anaplasmosis can cause fever and lethargy but the most notable feature is anemia due to phagocytosis of parasitized erythrocytes. Total costs of disease to U.S. cattle producers are difficult to estimate but are thought to amount to several hundred million dollars per annum. Treatment and control have historically been predicated on the administration of the antimicrobials oxytetracycline and chlortetracycline, respectively. In the U.S., disease prevention is complicated by the lack of efficacy data for the conditionally approved vaccine. In this dissertation, examples of the impact of bovine anaplasmosis on the domestic cattle population are explored. Two case reports are presented that describe the impact of disease outbreaks in areas considered both endemically stable and unstable. Two production systems and geographic regions were examined: a dairy in the state of Iowa and a beef cattle operation in Florida. Serology was performed in both cases using commercially available enzyme-linked immunosorbent linked assay (ELISA) kits. Outcome measures included seroprevalence of anti-Am antibodies, milk production, abortions, and mortality. In Iowa, seroprevalence of anti-Am antibodies was shown to have an inverse relationship with milk production. In Florida, seroprevalence of anti-Am antibodies was shown to be higher among open herds containing animals introduced from a different region (Texas). Likewise, open herds were shown to experience higher rates of mortality and abortion than herds containing exclusively Florida cattle. Statewide seroprevalence for Florida was also estimated by testing the blood of animals destined for slaughter. Seroprevalence among herds experiencing the disease outbreak was found to be higher than the statewide average. This dissertation also explores the efficacy of currently approved tetracycline-based treatment and control protocols. An experiment was conducted to study the impact of injectable oxytetracycline (OTC) and oral chlortetracycline (CTC) on Am bacteremia over time. Persistently infected cattle were treated with OTC, CTC, or no drug, and bacteremia was calculated using quantitative polymerase chain reaction (qPCR). Bacteremia was found to significantly but transiently drop among animals treated with OTC. Among those treated with CTC, no significant drop in bacteremia was noted. These findings are important as they show that neither drug, despite being approved by the FDA for treatment (OTC) and control (CTC) of bovine anaplasmosis, appears capable of bacterial clearance from persistently infected cattle. As vaccine options for bovine anaplasmosis are limited, practical implications of vaccine delivery via a subcutaneous implant are also explored. A series of pilot studies were conducted to test the capability of a subcutaneous implant to deliver vaccine over an extended period of time. For these pilots, a gonadotropin-releasing hormone (GnRH) antigen was used. To test the versatility of the implant, GnRH was bound to different carrier proteins (ovalbumin, OVA; keyhole limpet hemocyanin, KLH) and packaged with different adjuvants (Quil-A®, DEAE-Dextran). Outcome measures were related to the immunological impact of a GnRH antigen and included scrotal and testicular changes, levels of anti-GnRH antibodies, testosterone concentration, and degree of spermatogenesis. The implant was shown to be capable of stimulating humoral immunity to GnRH as determined by ELISA. The implant was also shown to affect spermatogenesis when gonads were examined histologically. Although significant changes in testosterone were not noted, the implant technology is promising. To apply the implantable vaccine concept to bovine anaplasmosis, a study was conducted to examine the ability of the implant to confer protection to heterologous disease challenge over a long period of time (20+ months). For this experiment, the major surface protein 1a (MSP1a), an adhesion conserved among many Am strains, was used as an antigen and both Quil-A® and DEAE-D were used as adjuvants. Outcome measures included bacteremia, body temperature, and packed cell volume (PCV) as an indicator of anemia. Animals that were provided vaccine implants with both adjuvants were shown to have reduced symptom severity after being challenged with Am. This is a promising outcome for the implant methodology and warrants further exploration. Bovine anaplasmosis remains a challenge to profitable cattle production in the U.S. and abroad. As tetracycline antimicrobials may not reliably clear infection, further research towards refining strategies of disease prevention is warranted. A vaccine that not only limits disease severity, but also entirely prevents infection with Am would be ideal. Novel implantable platforms for vaccine delivery are an attractive option, as they may be able to confer protection over an extended period of time with a single dose. Towards this end, field studies of an implantable polyvalent vaccine tailored to deliver highly-conserved Am surface epitopes are needed.