Delivering an Immunocastration Vaccine via a Novel Subcutaneous Implant.

Immunocastration relies on the vaccine-mediated stimulation of an immune response to gonadotropin-releasing hormone (GnRH) in order to interrupt spermatogenesis. This approach offers a less painful alternative to traditional castration approaches but the current, commercially available options require multiple doses of vaccine to maintain sterility. Thus, a series of pilot studies were conducted to determine the feasibility of a single-dose immunocastration vaccine implant. These five studies utilized a total of 44 Holstein bulls to determine the optimal vaccine composition and validate the ability of a stainless-steel subcutaneous implant to deliver a vaccine. Outcome measures included the duration of implant retention, scrotal dimensions and temperature, implant site temperature, anti-GnRH antibodies, and serum testosterone concentration. Over the course of several studies, anti-GnRH antibodies were successfully stimulated by vaccine implants. No significant treatment effects on scrotal dimensions or testosterone were detected over time, but changes in spermatogenesis were detected across treatment groups. Results indicate that a single-dose implantable immunocastration vaccine elicits a humoral immune response and could impact spermatogenesis in bulls. These findings provide opportunities for the refinement of this technology to improve implant retention over longer periods of time. Taken together, this approach will offer producers and veterinarians an alternative to physical castration methods, to improve animal welfare during routine livestock management procedures.

Characterization of a P-glycoprotein drug transporter from Toxocara canis with a novel pharmacological profile.

P-glycoproteins from the ATP-binding cassette transporter family are responsible for drug evasion by bacterial pathogens and neoplastic cells. More recently, these multidrug resistance transporters have been investigated for contributions to drug resistance in nematode parasites. In this study, we cloned and characterized the P-glycoprotein Tca-Pgp-11.1 from Toxocara canis, the canine intestinal ascarid. Large numbers of Tca-Pgp-11 transcripts were observed in the intestine of adult male and female worms. Heterologous expression studies confirmed sensitivity to known P-glycoprotein inhibitors. Interestingly, the competitive inhibitor verapamil had lower IC50 values than newer generation inhibitors that are designed to allosterically modulate mammalian P-glycoprotein. Consistent with other nematode P-glycoproteins, Tca-Pgp-11.1 was sensitive to ivermectin and selamectin but not moxidectin. Taken together, our data suggests that T. canis P-glycoproteins represent nematode-specific drug targets that could be exploited to enhance efficacy of existing anthelmintics.

Bovine immune response to leptospira antigen in different novel adjuvants and vaccine delivery platforms.

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P > 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P < 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle. Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock.

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.

Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis.

Brucellosis is a bacterial zoonosis and a significant source of economic loss and a major public health concern, worldwide. Bovine brucellosis, as caused primarily by Brucella abortus, is an important cause of reproductive loss in cattle. Vaccination has been the most effective way to reduce disease prevalence contributing to the success of control and eradication programs. Currently, there are no human vaccines available, and despite the success of commercial vaccines for livestock, such as B. abortus strain RB51 (RB51), there is need for development of novel and safer vaccines against brucellosis. In the current study, we report the fabrication of and immune responses to an implantable single dose polyanhydride-based, methanol-killed RB51 antigen containing delivery platform (VPEAR) in cattle. In contrast to animals vaccinated with RB51, we did not observe measurable RB51-specific IFN-γ or IgG responses in the peripheral blood, following initial vaccination with VPEAR. However, following a subsequent booster vaccination with RB51, we observed an anamnestic response in both vaccination treatments (VPEAR and live RB51). The magnitude and kinetics of CD4+ IFN-γ-mediated responses and circulating memory T cell subpopulations were comparable between the two vaccination treatments. Additionally, IgG titers were significantly increased in animals vaccinated with VPEAR as compared to live RB51- vaccinated animals. These data demonstrate that killed antigen may be utilized to generate and sustain memory, IFN-γ-mediated, CD4+ T cell and humoral responses against Brucella in a natural host. To our knowledge, this novel approach to vaccination against intracellular bacteria, such as Brucella, has not been reported before.

Use of praziquantel to control platyhelminth parasites of fish.

Fish are common definitive and intermediate hosts for a variety of parasitic flatworms. In unstressed wild populations, parasitic infections often go unnoticed and are perceived to represent a lesser threat to fish health. In contrast, platyhelminth parasitism of captive fish often results in decreased weight gain and increased mortality which often necessitates chemotherapeutic treatment. The presence of platyhelminth parasites in fish tissues is not only unappealing but in some cases also represents a threat to human health. In veterinary medicine, one of the most commonly used agents with anti-flatworm activity is praziquantel; yet, no praziquantel products are labeled for use in fish in the United States. Veterinarians may use praziquantel preparations approved for other vertebrate species under the Animal Medicinal Drug Use Clarification Act (AMDUCA). However, such extra-label use should be informed by scientific evidence including efficacy and tissue residue studies. Herein, we review studies testing the efficacy of praziquantel for treatment of platyhelminthes along with an assessment of routes of administration, pharmacokinetics, and toxicity information.

A single dose polyanhydride-based vaccine platform promotes and maintains anti-GnRH antibody titers.

Traditionally, vaccination strategies require an initial priming vaccination followed by an antigen boost to generate adequate immunity. Here we describe vaccination against a self-peptide for reproductive sterilization utilizing a three-stage vaccine platform consisting of gonadotropin releasing hormone multiple antigenic peptide (GnRH-MAP) as a soluble injection coupled with subcutaneous administration of polyanhydride-immobilized GnRH-MAP and a cyto-exclusive implant containing GnRH-MAP dendrimer-loaded polyanhydride. This strategy generated and maintained cell-mediated and humoral immunity for up to 41 weeks after a single vaccination in mice with enhanced antibody avidity over time. All intact implants had a grossly visible tissue interface with neovascularization and lymphocytic aggregates. Despite detectable immunity, sterility was not achieved and the immune response did not lead to azoospermia in male mice nor prevent estrus and ovulation in female mice. However, the vaccine delivery device is tunable and the immunogen, adjuvants and release rates can all be modified to enhance immunity. This technology has broad implications for the development of long-term vaccination schemes.

Efficacy of injectable praziquantel for elimination of trematode metacercariae in bluegills (Lepomis macrochirus) and quantification of parasite death by propidium iodide staining.

Digenean trematodes have complex life cycles and control of these flatworms can be accomplished by eliminating immature parasite stages from intermediate hosts. In aquaculture systems, presence of trematode metacercariae can negatively impact fish health and lead to economic losses. Posthodiplostomum minimum is a parasite of birds that uses bluegill sunfish (Lepomis macrochirus) as the intermediate host and is commonly found in fish used to stock waterways for recreational purposes. In this study, we evaluated killing of P. minimum metacercariae by injectable praziquantel in naturally infected bluegills. Using propidium iodide staining and motility assessment, we found that 5 mg/kg administered intramuscularly was effective for parasite killing. However, metacercarial death was not apparent until day 7 post-treatment. Our results demonstrated that propidium iodide staining is an effective method for detecting death in metacercariae recovered from treated fish. This method was at least as sensitive as objective motility scoring and provided quantitative assessment of parasite death. Future studies involving treatment of metacercariae in fish with praziquantel may need to be carried out over a period of weeks in order to accurately assess parasite killing and would benefit from using the propidium iodide method.