Pathogenesis and Host Responses to Viral Diseases in Livestock Species.

Infectious diseases in livestock species are responsible for significant economic losses worldwide and constantly threaten food security [...].

Virome Characterization in Commercial Bovine Serum Batches-A Potentially Needed Testing Strategy for Biological Products.

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies' established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.

Virome characterization in serum of healthy show pigs raised in Oklahoma demonstrated great diversity of ssDNA viruses.

In the United States, show pigs are raised to compete in agricultural events. These animals are usually raised in small herds with extensive human, domestic, and wild animal contact. Therefore, pathogen monitoring in this animal category is critical for improved disease surveillance and preparedness. This study characterized the virome of healthy show pigs using high-throughput sequencing using pooled serum samples from 2018 or 2019 (200 samples each pool). Results demonstrated the presence of DNA viral families (Parvoviridae, Circoviridae, and Herpesviridae) and RNA families (Arteriviridae, Flaviviridae, and Retroviridae). Twenty-three viral species were identified, including the first detection of porcine bufavirus in the US. Moreover, important swine pathogens identified included porcine reproductive and respiratory syndrome virus, atypical porcine pestivirus, and porcine circovirus (PCV). Additionally, complete coding genomes of 17 viruses from the Parvoviridae, Anelloviridae, and Circoviridae families were retrieved and included the first near full-length genomes of US Ungulate bocaparvovirus 3 species.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets.

Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.

Senecavirus A 3C Protease Mediates Host Cell Apoptosis Late in Infection.

Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3Cpro) in SVA-induced host-cell apoptosis and cleavage of NF-κB-p65. Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.

Pathogenicity and cross-reactive immune responses of a historical and a contemporary Senecavirus A strains in pigs.

The goals of this study were to compare the pathogenicity and infection dynamics of a historical and a contemporary SVA strains (SVV 001 and SD15-26) and to assess cross-neutralizing and cross-reactive T cell responses following experimental infection in pigs. Both SVA strains successfully infected all inoculated animals, resulting in viremia and robust antibody and cellular immune responses. SVA SD15-26 infection resulted in characteristic clinical signs and vesicular lesions, however, SVA SVV 001 did not cause overt clinical disease with inoculated animals remaining clinically normal during the experiment. Notably, neutralization- and -recall IFN-γ expression-assays revealed marked cross-neutralizing antibody and cross-reactive T cell responses between the two viral strains. Together these results demonstrate that the historical SVA SVV 001 strain presents low virulence in pigs when compared to the contemporary SVA SD15-26 strain. Additionally, immunological assays indicate that SVA SVV 001 and SD15-26 are antigenically related and share conserved antigenic determinants.

Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein.

Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.

Adaptive Immune Responses following Senecavirus A Infection in Pigs.

Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of αß T cells, especially CD4+ T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8+ and double-positive CD4+ CD8+ T cells (effector/memory T cells) expressing interferon gamma (IFN-γ) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host.IMPORTANCE Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4+ T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results.

BACKGROUND: Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. RESULTS: Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. CONCLUSIONS: Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

Early Detection of Circulating Antigen and IgM-Associated Immune Complexes during Experimental Mycobacterium bovis Infection in Cattle.

The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.

Increased TNF-α/IFN-γ/IL-2 and Decreased TNF-α/IFN-γ Production by Central Memory T Cells Are Associated with Protective Responses against Bovine Tuberculosis Following BCG Vaccination.

Central memory T cell (Tcm) and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector/memory populations from bacille Calmette-Guerin (BCG) vaccinated and non-vaccinated calves by flow cytometry prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves, only differing in magnitude (i.e., infected > vaccinated). BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (3 weeks post-infection), non-vaccinates had greater antigen-specific interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α) and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains) were detected in memory subsets, as well as in effector cells, as early as 3 weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden, while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB.

Interleukin-17A as a Biomarker for Bovine Tuberculosis.

T helper 17 (Th17)-associated cytokines are integral to the immune responses to tuberculosis, initiating both protective and harmful inflammatory responses. The aim of the present study was to evaluate applied aspects of interleukin-17 (IL-17) biology in the context of Mycobacterium bovis infection of cattle. Using transcriptome sequencing (RNA-Seq), numerous Th17-associated cytokine genes (including IL-17A, IL-17F, IL-22, IL-19, and IL-27) were upregulated >9-fold in response to purified protein derivative stimulation of peripheral blood mononuclear cells from experimentally M. bovis-infected cattle. Protective vaccines elicited IL-17A, IL-17F, IL-22, and IL-27 responses. Reduced IL-17A responses by vaccine recipients, compared to nonvaccinated animals, at 2.5 weeks after M. bovis challenge correlated with reduced disease burdens. Additionally, IL-17A and interferon gamma (IFN-γ) responses were highly correlated and exhibited similar diagnostic capacities. The present findings support the use of Th17-associated cytokines as biomarkers of infection and protection in the immune responses to bovine tuberculosis.

Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle.

Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-γ. Interferon-γ release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4(+) T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-γ ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle.

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

Relevance of bovine tuberculosis research to the understanding of human disease: historical perspectives, approaches, and immunologic mechanisms.

Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual benefit approach, demonstrating a profound relevance of veterinary studies for biomedical applications. Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is an exemplary model for the demonstration of this concept. Early studies with cattle were instrumental in the development of the use of Koch's tuberculin as an in vivo measure of cell-mediated immunity for diagnostic purposes. Calmette and Guerin demonstrated the efficacy of an attenuated M. bovis strain (BCG) in cattle prior to use of this vaccine in humans. The interferon-γ release assay, now widely used for TB diagnosis in humans, was developed circa 1990 for use in the Australian bovine TB eradication program. More recently, M. bovis infection and vaccine efficacy studies with cattle have demonstrated a correlation of vaccine-elicited T cell central memory (TCM) responses to vaccine efficacy, correlation of specific antibody to mycobacterial burden and lesion severity, and detection of antigen-specific IL-17 responses to vaccination and infection. Additionally, positive prognostic indicators of bovine TB vaccine efficacy (i.e., responses measured after infection) include: reduced antigen-specific IFN-γ, iNOS, IL-4, and MIP1-α responses; reduced antigen-specific expansion of CD4(+) T cells; and a diminished activation profile on T cells within antigen stimulated cultures. Delayed type hypersensitivity and IFN-γ responses correlate with infection but do not necessarily correlate with lesion severity whereas antibody responses generally correlate with lesion severity. Recently, serologic tests have emerged for the detection of tuberculous animals, particularly elephants, captive cervids, and camelids. B cell aggregates are consistently detected within tuberculous lesions of humans, cattle, mice and various other species, suggesting a role for B cells in the immunopathogenesis of TB. Comparative immunology studies including partnerships of researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in both man and animals.