Examining innate responses to Flaviviridae infection: how myeloid cells respond to and are modulated by bovine viral diarrhea virus in vivo and in vitro.

Disease associated with BVDV infection can be devastating ranging to millions of dollars in loss to the cattle industry. As well, infection with BVDV leads to an immune-suppressed host though an unknown mechanism(s). Although there are vaccines available, they are not broadly protective and ineffective in PI cattle. The myeloid lineage cells such as monocytes and MΦ are important in recognizing BVDV as well as initiating an adaptive immune response. There is little information on the effects of BVDV in vivo on the functionality of these cells and a better understanding can lead to potential treatment. Investigating the effects on cytokine production, expression and response of TLRs and functional aspects of phagocytosis are critical. The specific aims in this dissertation studies utilize neonatal calves for in vivo study and 1-year-old cattle for cell donors during in vitro investigation. BVDV-2 strain 1373 was utilized as it is a field strain, which induces severe acute disease and is a highly virulent. BVDV-2 strains RS886 and 28508 are subclinical acute disease inducing strains, which are lower in virulence and typical of field isolates from PI cattle or subclinical infections. BVDV-2 strain 296c is a cytopathic strain whereas BVDV-2 strain 296nc is an isogenic non-cytopathic strain. These isogenic strains were chosen as the only differences between the strains are the NS2/3 coding regions. BVDV-2 strains 1373 and RS886 were utilized for in vivo experimentation whereas BVDV-2 strains 1373, 28508, 296c and 296nc were used for in vitro experiments. Both in vivo and in vitro models were used to characterize viral effects on cytokine expression and secretion, TLR responsiveness, signaling events, and phagocytosis of myeloid cells after exposure to virus

Development of a bead-agglutination assay for rapid detection of Tritrichomonas foetus

Tritrichomonas foetus is a flagellated protozoan parasite that causes inflammation of the reproductive tract leading to early embryonic death and abortion in cattle, thereby resulting in significant economic losses. Testing and culling infected bulls is an important strategy for parasite control. Routine testing is mainly limited to bulls that are traveling across state lines or within states that have specific control programs. Both culture and PCR detection methods are available, but they are not typically conducted as part of a yearly breeding soundness program and are not easily conducted in the field. In the present study, we developed a bead agglutination assay for detection of T. foetus antigens. Our experiments revealed that latex beads conjugated to T. foetus lipophosphoglycan-binding antibodies visibly clump in the presence of T. foetus. The detection limit of the assay, determined using both field and laboratory isolates of the parasite, was 0.25 μg/mL and 1.0 μg/mL total T. foetus antigen, respectively. Our results indicate that an antigen detection test could offer a tool for screening bulls under field conditions

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

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

Examining innate responses to Flaviviridae infection: how myeloid cells respond to and are modulated by bovine viral diarrhea virus in vivo and in vitro.

Disease associated with BVDV infection can be devastating ranging to millions of dollars in loss to the cattle industry. As well, infection with BVDV leads to an immune-suppressed host though an unknown mechanism(s). Although there are vaccines available, they are not broadly protective and ineffective in PI cattle. The myeloid lineage cells such as monocytes and MΦ are important in recognizing BVDV as well as initiating an adaptive immune response. There is little information on the effects of BVDV in vivo on the functionality of these cells and a better understanding can lead to potential treatment. Investigating the effects on cytokine production, expression and response of TLRs and functional aspects of phagocytosis are critical. The specific aims in this dissertation studies utilize neonatal calves for in vivo study and 1-year-old cattle for cell donors during in vitro investigation. BVDV-2 strain 1373 was utilized as it is a field strain, which induces severe acute disease and is a highly virulent. BVDV-2 strains RS886 and 28508 are subclinical acute disease inducing strains, which are lower in virulence and typical of field isolates from PI cattle or subclinical infections. BVDV-2 strain 296c is a cytopathic strain whereas BVDV-2 strain 296nc is an isogenic non-cytopathic strain. These isogenic strains were chosen as the only differences between the strains are the NS2/3 coding regions. BVDV-2 strains 1373 and RS886 were utilized for in vivo experimentation whereas BVDV-2 strains 1373, 28508, 296c and 296nc were used for in vitro experiments. Both in vivo and in vitro models were used to characterize viral effects on cytokine expression and secretion, TLR responsiveness, signaling events, and phagocytosis of myeloid cells after exposure to virus.</p

Development of a bead-agglutination assay for rapid detection of Tritrichomonas foetus

Tritrichomonas foetus is a flagellated protozoan parasite that causes inflammation of the reproductive tract leading to early embryonic death and abortion in cattle, thereby resulting in significant economic losses. Testing and culling infected bulls is an important strategy for parasite control. Routine testing is mainly limited to bulls that are traveling across state lines or within states that have specific control programs. Both culture and PCR detection methods are available, but they are not typically conducted as part of a yearly breeding soundness program and are not easily conducted in the field. In the present study, we developed a bead agglutination assay for detection of T. foetus antigens. Our experiments revealed that latex beads conjugated to T. foetus lipophosphoglycan-binding antibodies visibly clump in the presence of T. foetus. The detection limit of the assay, determined using both field and laboratory isolates of the parasite, was 0.25 μg/mL and 1.0 μg/mL total T. foetus antigen, respectively. Our results indicate that an antigen detection test could offer a tool for screening bulls under field conditions.This article is published as Schaut, Robert G., Lynette B. Corbeil, Courtney N. Blake, and Matthew T. Brewer. "Development of a bead-agglutination assay for rapid detection of Tritrichomonas foetus." Veterinary Parasitology 243 (2017): 188-191. DOI: 10.1016/j.vetpar.2017.06.025.</p

Bovine Viral Diarrhea Virus Type 2 Impairs Macrophage Responsiveness to Toll-Like Receptor Ligation with the Exception of Toll-Like Receptor 7

<div><p><i>Bovine viral diarrhea virus</i> (BVDV) is a member of the <i>Flaviviridae</i> family. BVDV isolates are classified into two biotypes based on the development of cytopathic (cp) or non-cytopathic (ncp) effects in epithelial cell culture. BVDV isolates are further separated into species, BVDV1 and 2, based on genetic differences. Symptoms of BVDV infection range from subclinical to severe, depending on strain virulence, and may involve multiple organ systems and induction of a generalized immunosuppression. During BVDV-induced immune suppression, macrophages, critical to innate immunity, may have altered pathogen recognition receptor (PRR) signaling, including signaling through toll-like receptors (TLRs). Comparison of BVDV 2 strains with different biotypes and virulence levels is valuable to determining if there are differences in host macrophage cellular responses between viral phenotypes. The current study demonstrates that cytopathic (cp), noncytopathic (ncp), high (hv) or low virulence (lv) BVDV2 infection of bovine monocyte-derived macrophages (MDMΦ) result in differential expression of pro-inflammatory cytokines compared to uninfected MDMΦ. A hallmark of cp BVDV2 infection is IL-6 production. In response to TLR2 or 4 ligation, as might be observed during secondary bacterial infection, cytokine secretion was markedly decreased in BVDV2-infected MDMΦ, compared to non-infected MDMΦ. Macrophages were hyporesponsive to viral TLR3 or TLR8 ligation. However, TLR7 stimulation of BVDV2-infected MDMΦ induced cytokine secretion, unlike results observed for other TLRs. Together, these data suggest that BVDV2 infection modulated mRNA responses and induced a suppression of proinflammatory cytokine protein responses to TLR ligation in MDMΦ with the exception of TLR7 ligation. It is likely that there are distinct differences in TLR pathways modulated following BVDV2 infection, which have implications for macrophage responses to secondary infections.</p></div

Expression of proinflammatory cytokine gene transcription in MDMΦs inoculated with high and low virulence BVDV2 strains.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 with RNA harvested at 2, 6, 18, and 24 h after inoculation. Cytokine mRNA was analyzed by qPCR using ribosomal protein s9 (RPS9) as an endogenous control with fold change expressed relative to uninfected control MDMΦs harvested at the corresponding time points. <i>il1β</i> (A), <i>tnfα</i> (B), <i>il6</i> (C), <i>il8</i> (D), <i>il12p40</i> (E), <i>il10</i> (F) were measured using primer sets specific for bovine genes using SYRB Green chemistry. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. *** P < 0.001.</p

Proinflammatory cytokine secretion of BVDV2 inoculated or LPS stimulated MDMΦs 24 h after treatment.

<p>MDMΦs were differentiated in 96 well plates for 7 days and inoculated with BVDV2 strains in duplicate at an MOI of 1 or 2 μg/mL LPS with cell supernatants harvested at 24 h after treatment. Cytokine protein was analyzed by Searchlight Array using analytes specific for bovine IL-1β (A), IL-6 (B) and TNFα (C) with 50 μL of cell supernatant analyzed in duplicate. Cytokines were quantified by generation of standard curves against recombinant bovine cytokines provided by the manufacturer of the Searchlight platform. Bars represent the mean value ± SEM from four different experiments from 9 total donor cattle. ** P < 0.001.</p