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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus ), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30–40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3–4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

The bovine tuberculoid granuloma is the hallmark lesion of bovine tuberculosis (bTB) due to Mycobacterium bovis infection. The pathogenesis of bTB, and thereby the process of bovine tuberculoid granuloma development, involves the recruitment, activation, and maintenance of cells under the influence of antigen, cytokines and chemokines in affected lungs and regional lymph nodes. The granuloma is key to successful control of bTB by preventing pathogen dissemination through containment by cellular and fibrotic layers. Paradoxically, however, it may also provide a niche for bacterial replication. The morphologic and cellular characteristics of granulomas have been used to gauge disease severity in bTB pathogenesis and vaccine efficacy studies. As such, it is critical to understand the complex mechanisms behind granuloma initiation, development, and maintenance.

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.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis , continues to be a major economic burden associated with production losses and a public health concern due to its zoonotic nature. As with other intracellular pathogens, cell-mediated immunity plays an important role in the control of infection. Characterization of such responses is important for understanding the immune status of the host, and to identify mechanisms of protective immunity or immunopathology. This type of information can be important in the development of vaccination strategies, diagnostic assays, and in predicting protection or disease progression. However, the frequency of circulating M. bovis -specific T cells are often low, making the analysis of such responses difficult. As previously demonstrated in a different cattle infection model, antigenic expansion allows us to increase the frequency of antigen-specific T cells. Moreover, the concurrent assessment of cytokine production and proliferation provides a deeper understanding of the functional nature of these cells. The work presented here, analyzes the T cell response following experimental M. bovis infection in cattle via in vitro antigenic expansion and re-stimulation to characterize antigen-specific CD4, CD8, and γδ T cells and their functional phenotype, shedding light on the variable functional ability of these cells. Data gathered from these studies can help us better understand the cellular response to M. bovis infection and develop improved vaccines and diagnostic tools.

Background Pasteurella multocida is a Gram-negative coccobacillus and is the causative agent of fowl cholera in avian species. P. multocida expresses two large filamentous hemagglutinin (FhaB) proteins encoded by fhaB 1 and fhaB 2 genes. Previously, it was demonstrated that P. multocida FhaB2 is an important virulence factor in the development of fowl cholera disease. In the current study, we examined the potential role of FhaB1 in fowl cholera disease development. An fhaB1 deletion mutant, devoid of foreign DNA, was constructed using a temperature sensitive plasmid in a well-characterized P. multocida avian strain P-1059 (A:3). Results Real-time PCR assay confirmed the expression of full-length fhaB1 mRNA in the wild-type parent strain and truncated fhaB1 mRNA in the Δ fhaB1 mutant strain. Both parent and the mutant strain produced biofilm; however, the Δ fhaB1 mutant produced significantly lower amounts of biofilm. Turkey poults were challenged intranasally and intramuscularly to assess the virulence of the fhaB1 mutant and the wild-type parent strains. Contrary to our expectation, inactivation of fhaB1 did not reduce virulence by either challenge route. Conclusions These findings indicate that this large and highly conserved FhaB1 protein is not necessary for the development of acute fowl cholera disease in turkeys.

Background Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis , continues to be an animal and zoonotic concern in many parts of the world, including the United States. Long-standing eradication programs have been successful at lowering prevalence of disease in many countries; however, disease eradication has not been achieved. One major obstacle to eradication is the presence of various wildlife reservoirs for M. bovis , such as white-tailed deer ( Odocoileus virginianus ), which serve as a source of spill-back to cattle herds. A potential method to reduce intra- and inter-species disease transmission of M. bovis between wildlife and domestic livestock includes vaccination of wildlife species. Oral vaccination of white-tailed deer with the human tuberculosis vaccine, M. bovis bacillus Calmette-Guérin (BCG) has been demonstrated to afford some level of protection against experimental challenge. However, vaccinating wildlife presents its own challenges, primarily due to the need of a delivery platform that could be implemented at scale and would not require animal handling. Results Oral vaccine delivery units or baits are an effective means of delivering vaccine to wildlife populations. Therefore, we explored whether sodium alginate spheres could be used as a delivery platform for BCG for vaccination of white-tailed deer. We assessed the development of peripheral immune responses following BCG vaccination and demonstrated that passive administration of BCG via alginate spheres results in antigen-specific cellular responses, similar to oral administration of BCG. Conclusions Our data characterize the kinetics of cellular responses elicited by oral vaccination and suggest passive oral administration of BCG as a potential means to vaccinate free-ranging white-tailed deer.

Background Mycobacterium bovis BCG is the human tuberculosis vaccine and is the oldest vaccine still in use today with over 4 billion people vaccinated since 1921. The BCG vaccine has also been investigated experimentally in cattle and wildlife by various routes including oral and parenteral. Thus far, oral vaccination studies of cattle have involved liquid BCG or liquid BCG incorporated into a lipid matrix. Lyophilization is an established technique used for stabilizing bioproducts such as vaccines. Methods In the current study, cattle were vaccinated in two phases. In each phase, cattle were divided into three groups. Group 1 received BCG injected SQ, Group 2 received liquid BCG delivered to the posterior oral cavity, Group 3 orally consumed lyophilized BCG contained within a gelatin capsule placed within a small amount of a commercial alfalfa product. Results No vaccinated cattle were positive by an interferon gamma release assay. All but 4 animals were negative by tuberculin skin testing prior to vaccination: the 4 non-negative animals being categorized as suspects. Sixteen weeks post-vaccination all but 1 animal was negative, it being categorized as a suspect. An in vitro antigen stimulation assay and flow cytometry were used to detect antigen-specific CD4, CD8 and γδ T cell responses following vaccination. Oral vaccination of animals with lyophilized BCG did not result in any increases in the frequency of CD4, CD8 or γδ T cell proliferative or IFN-γ responses at any of the time points analyzed in either phase 1 or 2. In contrast, vaccination with BCG SQ and liquid BCG delivered to the posterior pharynx, resulted in an increase in the frequency of proliferating and IFN-γ-producing CD4 T cells with peak responses at 9–12 weeks post-vaccination. Similar to oral lyophilized BCG vaccinated animals, we did not observe any significant increases in the frequency of CD8 and γδ T cell proliferative and IFN-γ responses following SQ or oral liquid vaccinated animals. Conclusions These data would suggest that vaccination with oral lyophilized BCG does not induce a measurable, antigen-specific cell mediated responses in the periphery, when compared to BCG administered SQ or liquid BCG administered via the oral route. However, vaccination with either SQ or liquid BCG delivered to the posterior pharynx does induce measurable CD4 T cell responses in the periphery.

causes chronic respiratory disease with high mortality rates in American bison ( ). A recent study showed that a subunit vaccine containing elongation factor thermal unstable (EFTu) and heat shock protein 70 (Hsp70) antigens induced immunity and enhanced protection in bison, resulting in reduced lung lesions and bacterial loads following experimental challenge. This study aimed to characterize the transcriptional responses underlying this protection in vaccinated (  = 5) compared to unvaccinated control (  = 4) bison following infection. Two doses of vaccines were administered on day 0 and at 21 days post-vaccination (DPV), followed by intranasal inoculation with bovine herpesvirus-1 (BHV-1) at 36 DPV and at 40 DPV. RNA sequencing was performed on liver, palatine tonsil (PT), retropharyngeal lymph node (RPLN), tracheobronchial lymph node (TBLN), spleen, and whole blood samples. Blood was collected at 1st vaccination (Day 0), 2nd vaccination (21 days post-vaccination), BHV-1 inoculation (36 DPV), inoculation (40 DPV), and 1 week post inoculation (47 DPV). The greatest number of differentially expressed transcripts (DETs) (≤0.05 FDR) were found in blood at 36 DPV (123 total DETs) and in spleen (57 DETs). At 36 DPV, vaccinated animals showed upregulation of transcripts involved in in cell adhesion, T-helper cell (Th1/Th2/Th17) differentiation, and antigen processing and presentation. This signifies a robust response to the 2nd vaccine dose, which caused increased expression of , , and correlating to increased T cell proliferation. Notably, transcription factors and were upregulated in vaccinated animals. Spleen-specific regulation included transcripts involved in innate immune response, such as and . These findings highlight the robust immune response induced by the vaccine, particularly through T-cell mediated responses, demonstrating its potential to enhance protective immunity against in bison.

The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in Madin-Darby bovine kidney cells (MDBK) and 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. Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID , 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. Our immunoperoxidase staining, RT-qPCR, and TCID 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. Our 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.

Mycobacterium bovis is the cause of tuberculosis in most animals, most notably cattle. The stereotypical lesion of bovine tuberculosis is the granuloma; a distinct morphological lesion where host and pathogen interact and disease outcome (i.e., dissemination, confinement, or resolution) is determined. Accordingly, it is critical to understand host-pathogen interactions at the granuloma level. Host-pathogen interactions within individual granulomas at different stages of disease have not been examined in cattle. We examined bacterial burden and cytokine expression in individual pulmonary granulomas from steers at 30, 90, 180, and 270 days after experimental aerosol infection with M. bovis . Bacterial burdens within individual granulomas examined 30 days after infection were greater and more heterogenous (variable) than those examined 90 to 270 days after infection. Bacterial burdens did not correlate with expression of IFN-γ, TNF-α, TGF-β, granuloma stage, or lung lesion score, although there was a modest positive correlation with IL-10 expression. Granuloma stage did have modest positive and negative correlations with TNF-α and IL-10, respectively. Heterogeneity and mean expression of IFN-γ, IL-10 and TNF-α did not differ significantly over time, however, expression of TGF-β at 90 days was significantly greater than that seen at 30 days after infection.