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

An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 partially protects American bison from Mycoplasma bovis infection

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison

Antiviral activity of bovine type III interferon against bovine viral diarrhea virus is greatly reduced in bovine turbinate cells due to limited expression of IFN lambda receptor 1 (IL-28Rα)

IntroductionThe antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route.MethodsHere we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rβ that constitute the bovIFN-λ3 receptor.ResultsOur immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rβ subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rβ subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells.ConclusionOur results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells

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.This article is published as Boggiatto, Paola M., Robert G. Schaut, Carly Kanipe, Sean M. Kelly, Balaji Narasimhan, Douglas E. Jones, and Steven C. Olsen. "Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis." Heliyon 5, no. 8 (2019): e02370. DOI: 10.1016/j.heliyon.2019.e02370.</p

Pathology of the emerging Mycobacterium tuberculosis complex pathogen, M. mungi, in the banded mongoose (Mungos mungo)

Wild banded mongooses (Mungos mungo) in northeastern Botswana and northwest Zimbabwe are infected with a novel Mycobacterium tuberculosis complex (MTC) pathogen, Mycobacterium mungi. We evaluated gross and histologic lesions in 62 infected mongooses (1999–2017). Many tissues contained multifocal irregular, lymphohistiocytic to granulomatous infiltrates and/or multifocal or coalescing noncaseating to caseating granulomas with variable numbers of intralesional acid-fast bacilli. Over one-third of nasal turbinates examined had submucosal lymphohistiocytic to granulomatous infiltrates, erosion and ulceration of the nasal mucosa, bony remodeling, and nasal distortion. Similar inflammatory cell infiltrates expanded the dermis of the nasal planum with frequent ulceration. However, even in cases with intact epidermis, acid-fast bacilli were present in variable numbers among dermal infiltrates and on the epidermal surface among desquamated cells and debris, most commonly in small crevices or folds. In general, tissue involvement varied among cases but was highest in lymph nodes (50/54, 93%), liver (39/53, 74%), spleen (37/51, 73%), and anal glands/sacs (6/8, 75%). Pulmonary lesions were present in 67% of sampled mongooses (35/52) but only in advanced disseminated disease. The pathological presentation of M. mungi in the banded mongoose is consistent with pathogen shedding occurring through scent-marking behaviors (urine and anal gland secretions) with new infections arising from contact with these contaminated olfactory secretions and percutaneous movement of the pathogen through breaks in the skin, nasal planum, and/or skin of the snout. Given the character and distribution of lesions and the presence of intracellular acid-fast bacilli, we hypothesize that pathogen spread occurs within the body through a hematogenous and/or lymphatic route. Features of prototypical granulomas such as multinucleated giant cells and peripheral fibrosis were rarely present in affected mongooses. Acid-fast bacilli were consistently found intracellularly, even in regions of necrosis. The mongoose genome has a unique deletion (RD1mon) that includes part of the encoding region for PPE68 (Rv3873), a gene co-operonic with PE35. These proteins can influence the host’s cellular immune response to mycobacterial infections, and it remains uncertain how this deletion might contribute to observed patterns of pathology. M. mungi infection in banded mongooses is characterized by both a unique transmission and exposure route, as well as accompanying pathological features, providing an opportunity to increase our understanding of MTC pathogenesis across host-pathogen systems.This work was supported in part by the Morris Animal Foundation (grant D14ZO-083) and the National Science Foundation (grant 1518663) as part of the joint NSF-NIH-USDA Ecology and Evolution of Infec-tious Diseases program. K. A. Alexander was also supported in part through the National Institutes of Health and National Institute of General Medical Sciences—Models of Infectious Disease Agent Study (grant 5U01GM070694-13).http://journals.sagepub.com/home/vethj2018Paraclinical Science

Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis)

Abstract Background Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. Results The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. Conclusions The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations

Additional file 1 of Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis)

Supplementary Material