An integrative and multi-indicator approach for wildlife health applied to an endangered caribou herd.

Assessing wildlife health in remote regions requires a multi-faceted approach, which commonly involves convenient samplings and the need of identifying and targeting relevant and informative indicators. We applied a novel wildlife health framework and critically assessed the value of different indicators for understanding the health status and trends of an endangered tundra caribou population. Samples and data from the Dolphin and Union caribou herd were obtained between 2015 and 2021, from community-based surveillance programs and from captured animals. We documented and categorized indicators into health determinants (infectious diseases and trace elements), processes (cortisol, pathology), and health outcomes (pregnancy and body condition). During a recent period of steep population decline, our results indicated a relatively good body condition and pregnancy rates, and decreasing levels of stress, along with a low adult cow survival. We detected multiple factors as potential contributors to the reduced survival, including Brucella suis biovar 4, Erysipelothrix rhusiopathiae and lower hair trace minerals. These results remark the need of targeted studies to improve detection and investigations on caribou mortalities. We also identified differences in health indicators between captured and hunter sampled caribou, highlighting the importance of accounting for sampling biases. This integrative approach that drew on multiple data sources has provided unprecedented knowledge on the health in this herd and highlights the value of documenting individual animal health to understand causes of wildlife declines.

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens.

Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

Expression and role of VLA-1 in resident memory CD8 T cell responses to respiratory mucosal viral-vectored immunization against tuberculosis.

Lung resident memory T cells (TRM) characterized by selective expression of mucosal integrins VLA-1 (α1ß1) and CD103 (αEß7) are generated following primary respiratory viral infections. Despite recent progress, the generation of lung TRM and the role of mucosal integrins following viral vector respiratory mucosal immunization still remains poorly understood. Here by using a replication-defective viral vector tuberculosis vaccine, we show that lung Ag-specific CD8 T cells express both VLA-1 and CD103 following respiratory mucosal immunization. However, VLA-1 and CD103 are acquired in differential tissue sites with the former acquired during T cell priming in the draining lymph nodes and the latter acquired after T cells entered the lung. Once in the lung, Ag-specific CD8 T cells continue to express VLA-1 at high levels through the effector/expansion, contraction, and memory phases of T cell responses. Using a functional VLA-1 blocking mAb, we show that VLA-1 is not required for trafficking of these cells to the lung, but it negatively regulates them in the contraction phase. Furthermore, VLA-1 plays a negligible role in the maintenance of these cells in the lung. Our study provides new information on vaccine-inducible lung TRM and shall help develop effective viral vector respiratory mucosal tuberculosis vaccination strategies.

A human type 5 adenovirus-based tuberculosis vaccine induces robust T cell responses in humans despite preexisting anti-adenovirus immunity.

There is an urgent need to develop new tuberculosis (TB) vaccines to safely and effectively boost Bacille Calmette-Guérin (BCG)-triggered T cell immunity in humans. AdHu5Ag85A is a recombinant human type 5 adenovirus (AdHu5)-based TB vaccine with demonstrated efficacy in a number of animal species, yet it remains to be translated to human applications. In this phase 1 study, we evaluated the safety and immunogenicity of AdHu5Ag85A in both BCG-naïve and previously BCG-immunized healthy adults. Intramuscular immunization of AdHu5Ag85A was safe and well tolerated in both trial volunteer groups. Moreover, although AdHu5Ag85A was immunogenic in both trial volunteer groups, it much more potently boosted polyfunctional CD4(+) and CD8(+) T cell immunity in previously BCG-vaccinated volunteers. Furthermore, despite prevalent preexisting anti-AdHu5 humoral immunity in most of the trial volunteers, we found little evidence that such preexisting anti-AdHu5 immunity significantly dampened the potency of AdHu5Ag85A vaccine. This study supports further clinical investigations of the AdHu5Ag85A vaccine for human applications. It also suggests that the widely perceived negative effect of preexisting anti-AdHu5 immunity may not be universally applied to all AdHu5-based vaccines against different types of human pathogens.

A toll-like receptor 3 ligand enhances protective effects of vaccination against Marek's disease virus and hinders tumor development in chickens.

Marek's disease (MD) is caused by Marek's disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

Regulation of TB vaccine-induced airway luminal T cells by respiratory exposure to endotoxin.

Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained.

A novel genetically engineered Mycobacterium smegmatis-based vaccine promotes anti-TB immunity.

Pulmonary TB remains a global health threat. Prophylactic immunization with Mycobacterium bovis BCG is the only key strategy to control TB. Ineffectiveness of BCG immunization in TB-endemic areas and BCG-related safety issues in HIV-positive infants have prompted the development of new TB vaccines. As enhanced understanding of the immune evasion mechanism of Mycobacterium tuberculosis will help develop new vaccines, Sweeney et al. studied the role of the esx-3 locus in mycobacterial pathogenesis. They have identified a previously unappreciated function of the esx-3 locus in innate immune evasion. They further discovered that Mycobacterium smegmatis with the esx-3 genes deleted could function as a novel vaccine vector with an enhanced innate immune-activating property. This vector, when engineered to express M. tuberculosis esx-3, was found to be a potent TB vaccine capable of a level of protection superior to that of BCG when administered via the intravenous route.

Transcriptome and proteome profiling of host responses to Marek's disease virus in chickens.

Marek's disease (MD) is an immunosuppressive and proliferative disease of domestic chickens caused by a highly oncogenic cell-associated alpha-herpesvirus, named Marek's disease virus (MDV). Despite the availability of highly efficacious vaccines for control of MD and existence of lines of chickens which display differential genetic susceptibility or resistance to this disease, little is known about the underlying mechanisms of MDV-host interactions. The recent advent of global or targeted gene and protein expression profiling has paved the way towards gaining a better understanding of host responses to MDV. The main objective of this review is to discuss some of the recent advancements made in relation to elucidating the mechanisms of MDV pathogenesis, host responses to MDV, genetic resistance/susceptibility to MD, and immunity conferred by vaccines. In this regard, particular emphasis has been placed on studies employing proteome and transcriptome profiling approaches. Finally, the utility of microRNA and RNA interference (RNAi) technologies for functional analysis of genes, proteins, and pathways that play a role in the complex interactions between MDV and its host is discussed.

Immune responses against Marek's disease virus.

It is more than a century since Marek's disease (MD) was first reported in chickens and since then there have been concerted efforts to better understand this disease, its causative agent and various approaches for control of this disease. Recently, there have been several outbreaks of the disease in various regions, due to the evolving nature of MD virus (MDV), which necessitates the implementation of improved prophylactic approaches. It is therefore essential to better understand the interactions between chickens and the virus. The chicken immune system is directly involved in controlling the entry and the spread of the virus. It employs two distinct but interrelated mechanisms to tackle viral invasion. Innate defense mechanisms comprise secretion of soluble factors as well as cells such as macrophages and natural killer cells as the first line of defense. These innate responses provide the adaptive arm of the immune system including antibody- and cell-mediated immune responses to be tailored more specifically against MDV. In addition to the immune system, genetic and epigenetic mechanisms contribute to the outcome of MDV infection in chickens. This review discusses our current understanding of immune responses elicited against MDV and genetic factors that contribute to the nature of the response.

Vaccine-induced host responses against very virulent Marek's disease virus infection in the lungs of chickens.

The aim of this study was to investigate the kinetics of virus replication and cellular responses in the lungs following infection with Marek's disease virus (MDV) and/or vaccination with herpesvirus of turkey (HVT) via the respiratory route. Chickens vaccinated with HVT and challenged with MDV had a higher accumulation of MDV and HVT genomes in the lungs compared to the chickens that received HVT or MDV alone. This increase in virus load was associated with augmented expression of interferon (IFN)-gamma and interleukin (IL)-10, and increased T cell infiltration. These findings shed more light on the immunological events that occur in the lungs after vaccination or infection with MDV.

Marek's disease virus influences the expression of genes associated with IFN-gamma-inducible MHC class II expression.

Chickens infected with Marek's disease virus (MDV) become lifelong carriers regardless of their susceptibility to clinical disease. Therefore various viral immune-evasive mechanisms must play a role in MDV-host interactions. MDV has previously been shown to influence the expression of major histocompatibility complex (MHC) class II molecules. However, little is known about the underlying mechanisms of this phenomenon. In the present study, we studied the effect of MDV infection on the expression of several genes associated with IFN-gamma-inducible MHC class II expression at 4, 7, 14, and 21 days post-infection (dpi). There was a significant (p

Proteomic analysis of host responses to Marek's disease virus infection in spleens of genetically resistant and susceptible chickens.

Resistance to Marek's disease (MD) in chickens is genetically regulated and there are lines of chickens with differential susceptibility or resistance to this disease. The present study was designed to study comparative changes in the spleen proteomes of MD-susceptible B19 and MD-resistant B21 chickens in response to MDV infection. Spleen proteomes were examined at 4, 7, 14 and 21 days post-infection (d.p.i.) using two-dimensional gel electrophoresis and subsequently the protein spots were identified by one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). On average, there were 520+/-27 distinct protein spots on each gel and 1.6+/-0.7% of the spots differed quantitatively in their expression (p< or =0.05 and fold change > or =2) between infected B19 and B21 chickens. There was one spot at 4d.p.i. and three spots each at the rest of the time points, which had a qualitative difference in expression. Most of the differentially expressed proteins at 4 and 7d.p.i. displayed increased expression in B21 chickens; conversely the differentially expressed proteins at 14 and 21d.p.i. showed an increase in expression in B19 chickens. The differentially expressed proteins identified in the present study included antioxidants, molecular chaperones, proteins involved in the formation of cytoskeleton, protein degradation and antigen presentation, signal transduction, protein translation and elongation, RNA processing and cell proliferation. These findings shed light on some of the underlying processes of genetic resistance or susceptibility to MD.

Analyses of the spleen proteome of chickens infected with Marek's disease virus.

Marek's disease virus (MDV), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. In the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to MDV infection were studied using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). Comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. In total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (FDR)or=2) or qualitatively differentially expressed at least once during different sampling points. Overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin-proteasome protein degradation (UPP), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. Notably, early stages of the disease were characterized by changes in the UPP, and antigen presentation. Furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. The present findings provide a basis for further studies aimed at elucidation of the role of these proteins in MDV interactions with its host.

Marek's disease virus-induced expression of cytokine genes in feathers of genetically defined chickens.

Marek's disease (MD) vaccines, although effective in reducing lymphoproliferation, cannot control infectious virus production in the feather follicle epithelium (FFE) which is the site of virus shedding. Therefore, we investigated Marek's disease virus (MDV) replication as well as the expression of cytokine genes in feathers of MDV-infected chickens belonging to genetically defined lines (N2a or B(21)/B(21) haplotype-resistant and P2a or B(19)/B(19) haplotype-susceptible). Though there was not a difference in MDV genome load and transcripts between feathers of these chicken lines at 4 and 10 days post-infection (d.p.i.), feathers of resistant chickens carried significantly lower viral genome load and transcripts at 21 d.p.i. Irrespective of genetic background of the chickens examined, MDV replication showed a significant positive correlation with the expression of IFN-gamma gene. The results imply the usefulness of genetic control approach in reducing virulent MDV transmission.

Cytokine gene expression patterns associated with immunization against Marek's disease in chickens.

The present study explored the immunological correlates of protection mediated by a live bivalent vaccine consisting of herpesvirus of turkeys (HVT) and SB-1 against infection with the RB1B strain of Marek's disease virus (MDV). Compared to unvaccinated infected chickens, vaccinated protected birds had lower expression of interleukin (IL)-6, IL-10 and IL-18 genes in spleen. However, there was no difference between these two groups of birds in the expression of interferon (IFN)-gamma, IL-4, IL-12 and inducible nitric oxide synthase (iNOS) genes on day 21 post-infection. Furthermore, protection was associated with lower MDV genome load in spleen but not in feather tips, suggesting that vaccination had little or no effect on curtailing virus transmission. In conclusion, vaccination with a bivalent MD vaccine was associated with distinct cytokine expression patterns in spleen and modulation of cytokine responses by the vaccine may play a role in mediation of protection.