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Hamartomas are benign tumor-like lesions composed of disorganized growth of mature mesenchymal or epithelial tissues indigenous to the organ involved. Sporadically observed in ruminants, vascular, fibrous, nasal, and pulmonary hamartomas have been reported in calves; pulmonary and cutaneous forms have been reported in sheep. A full-term elk calf found dead had a large intrathoracic mass replacing the left caudal lung lobe and compressing other thoracic organs. Histologically, cross- and tangential sections of bronchi were separated by collagenous mesenchyme and irregularly shaped canaliculi and saccules resembling terminal bronchioles. Rarely present were regions in which saccules, lined by simple cuboidal epithelium, transitioned into attenuated epithelium lining fully developed alveoli. These findings are consistent with a pulmonary hamartoma. To our knowledge, pulmonary hamartoma has not been reported previously in a non-domestic ruminant.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4–8 wk), late vaccination (21–28 wk) or booster vaccination, early after-experimental challenge (2–4 wk), and late after-experimental challenge (8–21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine ( n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase ( p < 0.05) delta millipolarization (mP; delta mP = sample mP − negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus.

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle. Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock.

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 rates approaching 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 and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2, WTD fawns 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. Infectious virus was recovered from 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.Author summaryThe high susceptibility of white-tailed deer (WTD) to SARS-CoV-2, their ability to transmit the virus to other deer, and the recent findings suggesting widespread SARS-CoV-2 infection in wild WTD populations in the U.S. underscore the need for a better understanding of the infection and transmission dynamics of SARS-CoV-2 in this potential reservoir species. Here we investigated the transmission dynamics of SARS-CoV-2 over time and defined the major sites of virus replication during the acute phase of infection. Additionally, we assessed the evolution of the virus as it replicated and transmitted between animals. The work provides important information on the infection dynamics of SARS-CoV-2 in WTD, an animal species that - if confirmed as a new reservoir of infection - may provide many opportunities for exposure and potential zoonotic transmission of the virus back to humans.

Bison (Bison bison) heifer calves (n = 32) were randomly assigned to control or vaccination with 1010 colony-forming units of Brucella abortus strain RB51 (RB51) vaccine by single or boostered parenteral delivery, or by surgical implantation of a dry dart formulation (n = 8/trt). Serum and/or peripheral blood mononuclear cells (PBMC) were obtained at 0, 4, 8, 13, 16, 21, and 24 wks after initial vaccination and at 0, 4, 8, 12, 15, 22, and 27 wks after booster vaccination to characterize humoral and cellular immune responses to RB51. Bison in both RB51 vaccination treatments demonstrated greater (P < 0.0001) serum humoral responses when compared to non-vaccinates, with parenteral vaccinates demonstrating greater (P < 0.01) responses when compared to mean responses of bison inoculated with the dry dart. Only the booster vaccinated treatment demonstrated greater (P < 0.0001) humoral responses than control bison in samples collected after re-inoculation. At 4, 8, 12, 16, and 24 wks after initial vaccination, PBMC from parenteral RB51 vaccinates demonstrated greater proliferative responses to RB51 when compared to responses of control animals. In comparison, bison inoculated with the RB51 dry dart did not demonstrate greater (P > 0.05) proliferative responses when compared to responses of non-vaccinates. Bison were pasture bred and pregnant animals experimentally challenged in mid-gestation with 107 CFU of B. abortus strain 2,308. Bison in parenteral vaccination treatments had reduced (P < 0.05) abortions and infection in uterine and fetal samples as compared to non-vaccinated bison, with booster vaccinates tending to have the lowest colonization (CFU/gm) in tissues. In comparison, the dry dart formulation did reduce abortion (P < 0.05) but not infection (P > 0.05) in most tissues when compared to non-vaccinated bison. The results of this study reaffirm the efficacy of boostered parenteral vaccination of bison with RB51 in preventing brucellosis. Our data also suggests that the novel dry dart RB51 formulation does not induce sufficient efficacy in bison after a single inoculation.

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.

Cattle are natural hosts of the intracellular pathogen Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of uniquely barcoded B. abortus strains to temporally and spatially quantify population structure during colonization of cattle through a natural route of infection. Introducing 10(8) bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a 1-wk time point. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached a parotid lymph node. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present at 1 wk postinfection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues.

Cattle are natural hosts of the intracellular pathogen, Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of approximately 106 uniquely barcoded B. abortus strains to temporally and spatially quantify population structure at the strain level during colonization of cattle through a natural route of infection. Introducing 108 bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a one-week timepoint. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached the parotid lymph nodes. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present one-week post infection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues.Significance statementUnderstanding microbial population dynamics during infection has important implications for disease management, transmission and pathogen evolution. A quantitative analysis of microbial population structure requires the ability to track individual strains. We used a pool of individually barcoded strains to measure changes in Brucella abortus population structure during infection of bovine hosts via the ocular conjunctiva, a natural route of entry. Cattle exert a severe bottleneck on the bacterial population entering through the conjunctival mucosa such that individual cells have a 0.0001 probability of colonizing a local draining lymph node. The populations in lymph nodes, even on different sides of the same animal, are distinct and dominated by a small number of highly abundant, spatially distinct clones.

Feral swine (Sus scrofa) are a destructive invasive species widespread throughout the United States that disrupt ecosystems, damage crops, and carry pathogens of concern for the health of domestic stock and humans includingBrucella suis—the causative organism for swine brucellosis. In domestic swine, brucellosis results in reproductive failure due to abortions and infertility. Contact with infected feral swine poses spillover risks to domestic pigs as well as humans, companion animals, wildlife, and other livestock. Genetic factors influence the outcome of infectious diseases; therefore, genome wide association studies (GWAS) of differential immune responses among feral swine can provide an understanding of disease dynamics and inform management to prevent the spillover of brucellosis from feral swine to domestic pigs. We sought to identify loci associated with differential antibody responses among feral swine naturally infected withB. suisusing a case-control GWAS. Tissue, serum, and genotype data (68,516 bi-allelic single nucleotide polymorphisms) collected from 47 feral swine were analyzed in this study. The 47 feral swine were culture positive forBrucellaspp. Of these 47, 16 were antibody positive (cases) whereas 31 were antibody negative (controls). Single-locus GWAS were performed using efficient mixed-model association eXpedited (EMMAX) methodology with three genetic models: additive, dominant, and recessive. Eight loci associated with seroconversion were identified on chromosome 4, 8, 9, 10, 12, and 18. Subsequent bioinformatic analyses revealed nine putative candidate genes related to immune function, most notably phagocytosis and induction of an inflammatory response. Identified loci and putative candidate genes may play an important role in host immune responses toB. suisinfection, characterized by a detectable bacterial presence yet a differential antibody response. Given that antibody tests are used to evaluate brucellosis infection in domestic pigs and for disease surveillance in invasive feral swine, additional studies are needed to fully understand the genetic component of the response toB. suisinfection and to more effectively translate estimates ofBrucellaspp. antibody prevalence among feral swine to disease control management action.

Rocky Mountain elk (Cervus canadensis) is a major reservoir for Brucella abortus in the Greater Yellowstone area, which has significant economic implications to the cattle industry. Vaccination attempts against intracellular bacterial diseases in elk populations have not been successful due to a negligible adaptive cellular immune response. A lack of genomic resources has impeded attempts to better understand why vaccination does not induce protective immunity. To overcome this limitation, PacBio, Illumina, and HiC sequencing with a total of 686-fold coverage was used to assemble the elk genome into 35 pseudomolecules. A robust gene annotation was generated resulting in 18,013 gene models and 33,422 mRNAs. The accuracy of the assembly was assessed using synteny to the red deer and cattle genomes identifying several chromosomal rearrangements, fusions and fissions. Because this genome assembly and annotation provide a foundation for genome-enabled exploration of Cervus species, we demonstrate its utility by exploring the conservation of immune system-related genes. We conclude by comparing cattle immune system-related genes to the elk genome, revealing nine putative gene losses in elk.Author SummaryBrucellosis, also known as contagious abortion, is a bacterial disease that commonly affects livestock and remains prevalent in Rocky Mountain elk (Cervus canadensis). Since the 1920’s the USDA has led a program to eradicate Brucellosis from cattle, yet wild Rocky Mountain elk continue to be a source of transmission. Attempts to vaccinate wild elk herds have been unsuccessful, due to a poor and short-lived immune response. To investigate the genetic basis for this inherent difference, we created the first genome and annotation for the Rocky Mountain elk. This genome assembly is of the highest quality and contains single linear sequences for all 35 chromosomes. In order to generate gene models, an array of RNA-Seq data and proteins from many different organ tissues and cells were used in gene prediction software. Specifically, we compare cattle immune system genes with the Rocky Mountain elk, revealing the putative loss of nine immune-system related genes in elk.

Vaccines are one of the most important tools available to prevent and reduce the incidence of infectious diseases in cattle. Despite their availability and widespread use to combat many important pathogens impacting cattle, several of these products demonstrate variable efficacy and safety in the field, require multiple doses, or are unstable under field conditions. Recently, nanoparticle-based vaccine platforms (nanovaccines) have emerged as promising alternatives to more traditional vaccine platforms. In particular, polymer-based nanovaccines provide sustained release of antigen payloads, stabilize such payloads, and induce enhanced antibod- and cell-mediated immune responses, both systemically and locally. To improve vaccine administrative strategies and efficacy, they can be formulated to contain multiple antigenic payloads and have the ability to protect fragile proteins from degradation. Nanovaccines are also stable at room temperature, minimizing the need for cold chain storage. Nanoparticle platforms can be synthesized for targeted delivery through intranasal, aerosol, or oral administration to induce desired mucosal immunity. In recent years, several nanovaccine platforms have emerged, based on biodegradable and biocompatible polymers, liposomes, and virus-like particles. While most nanovaccine candidates have not yet advanced beyond testing in rodent models, a growing number have shown promise for use against cattle infectious diseases. This review will highlight recent advancements in polymeric nanovaccine development and the mechanisms by which nanovaccines may interact with the bovine immune system. We will also discuss the positive implications of nanovaccines use for combating several important viral and bacterial disease syndromes and consider important future directions for nanovaccine development in beef and dairy cattle.

Bovine brucellosis, cause by infection with Brucella abortus, causes reproductive failure in cattle, has a major economic impact to producers, and as a zoonoses, it is a disease of public health concern. Characterization of the protective immune response against Brucella infection is important to our understanding of disease pathogenesis and for the development of diagnostic assays and vaccines. Most of the knowledge regarding protection against Brucella comes from studies in the murine model, but less is known about the immune responses in cattle. Assessment of antigen-specific T cell frequency and functional phenotype are critical to understand the immune status of the host, characterize mechanisms of protective immunity and immunopathology, and to predict immune protection. The frequency of circulating T cells specific for a particular pathogen is often very low, making analysis of such responses difficult. Our goal was to develop a flow-cytometry based approach to better track Brucella-specific T cell responses. Using peripheral blood mononuclear cells (PMBC) from Brucella abortus strain RB51-vaccinated cattle, we optimized an in vitro stimulation protocol based on a combination of antigen and pan-T cell stimulation. We then assessed RB51-specific T cell responses by concurrently measuring proliferation and cytokine production using flow-cytometry. This methodology enhances the detection of peripheral, Brucella-specific responses in cattle following RB51 vaccination. This protocol is versatile in that it can be modified to fit other in vitro stimulation systems and additional functional or phenotypic parameters can be added for flow cytometric detection and characterization of antigen-specific T cells.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.

Background Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. Results Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. Conclusions Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems. Electronic supplementary material The online version of this article (10.1186/s12867-018-0111-x) contains supplementary material, which is available to authorized users.

The inflammasome serves as a mechanism by which the body senses damage or danger. These multiprotein complexes form in the cytosol of myeloid, epithelial and potentially other cell types to drive caspase-1 cleavage and the secretion of the pro-inflammatory cytokines IL-1β and IL-18. Different types of inflammasomes, centered on (and named after) their cytosolic NLRs, respond to signals from bacteria, fungi, and viruses, as well as "sterile inflammatory" triggers. Despite the large body of research accumulated on rodent and human inflammasomes over the past 15 years, only recently have studies expanded to consider the role of inflammasomes in veterinary and wildlife species. Due to the key role of inflammasomes in mediating inflammatory responses observed in humans and rodents, characterization of the similarities and differences between humans/rodents and veterinary species is required to identify genetic and evolutionary influences on disease responses and to develop therapeutic candidates for use in veterinary inflammatory syndromes. Here, we summarize recent findings on inflammasomes in swine, cattle, dogs, bats, small ruminants, and birds. We describe current gaps in our knowledge and highlight promising areas for future research.

Three virulent species of Brucella are currently listed as biological select agent and toxins (BSAT) in the United States. During recent biannual reviews of listed agents and toxins, these virulent...

In response to several publicized failures of inactivation procedures, the Select Agent program in the United States has recently focused on validation or documentation of inactivation methods appr...

Inflammasomes act as sensors of infection or damage to initiate immune responses. While extensively studied in rodents, understanding of livestock inflammasomes is limited. The NLRP1 inflammasome sensor in rodents is activated by Toxoplasma gondii, Bacillus anthracis lethal toxin (LT), and potentially other zoonotic pathogens. LT activates NLRP1 by N-terminal proteolysis, inducing macrophage pyroptosis and a pro-inflammatory cytokine response. In contrast, NLRP1 in macrophages from humans and certain rodent strains is resistant to LT cleavage, and pyroptosis is not induced. Evolution of NLRP1 sequences towards those leading to pyroptosis is of interest in understanding innate immune responses in different hosts. We characterized NLRP1 in cattle ( Bos taurus) and American bison ( Bison bison). Bovine NLRP1 is not cleaved by LT, and cattle and bison macrophages do not undergo toxin-induced pyroptosis. Additionally, we found a predicted Nlrp1 splicing isoform in cattle macrophages lacking the N-terminal domain. Resistance to LT in bovine and human NLRP1 correlates with evolutionary sequence similarity to rodents. Consistent with LT-resistant rodents, bovine macrophages undergo a slower non-pyroptotic death in the presence of LPS and LT. Overall, our findings support the model that NLRP1 activation by LT requires N-terminal cleavage, and provide novel information on mechanisms underlying immune response diversity.

Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2−) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.

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 in-depth understanding of the interactions of vaccine delivery vehicles with antigen presenting cells is important for tailoring optimal adjuvant properties. Polymeric nanoparticles have been widely studied as adjuvants and delivery vehicles; however, there is little information regarding the effect of serum protein adsorption onto biomaterials and the effect of this adsorption upon interactions with antigen presenting cells. The current studies analyzed effects of polyanhydride chemistry on serum adsorption to nanoparticles with respect to their uptake by and activation of bone marrow-derived macrophages. Differential effects of serum adsorption based on nanoparticle chemistry were shown to enhance (for 1,6-bis(p-carboxyphenoxy)hexane and sebacic anhydride-based) or reduce (for 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane-based) nanoparticle uptake. The observed complex interdependence between nanoparticle chemistry and serum protein adsorption on macrophage activation...

To assess the effects of challenge dose and stage of gestation on infection and abortion, 35 elk were conjunctivally challenged with virulent Brucella abortus strain 2308 (S2308) during pregnancy. Seventeen elk were experimentally challenged early in the second trimester of gestation (December) with high (approximately 108 CFU) or low challenge (approximately 107 CFU) treatments having 8 and 9 pregnant elk, respectively. Other pregnant elk were experimentally challenged at a later challenge time (approximately early third trimester, February), with high and low challenge treatments having 8 and 10 elk, respectively. Conjunctival swabs from all animals were culture positive for the S2308 strain at 7 days after experimental challenge. All animals seroconverted on a B. abortus ELISA but optical density readings were not influenced (P > 0.05) by time of challenge or by challenge dosage. In the early challenge group, abortions occurred in 2 of 9 (22%) in the low challenge treatment and 3 of 8 (37%) in the high challenge treatment, whereas in the later challenge group, 1 of 8 (12.5%) in the low challenge treatment and 2 of 10 (20%) in the high challenge treatment aborted. The ability to recover B. abortus from samples obtained at necropsy did not differ (P > 0.05) between early and late challenges or between high and low challenge treatments. Despite the lack of abortions observed after experimental challenge, recovery from maternal tissues ranged from 50% (low dose, late challenge) to 77% (low dose, early challenge). Our data suggests that naive elk do not abort as frequently after experimental infection with B. abortus strain 2308 as compared to similar data in cattle and bison.

Brucellosis in sheep and goats, a zoonotic disease primarily associated with Brucella melitensis infections, causes significant economic losses and public health concerns worldwide. Although control measures are effective, economic limitations and nomadic lifestyles may limit vaccination coverage, and test and removal policies may not be feasible. In this study, we evaluated the effects of therapy with a long acting antimicrobial tulathromycin on the pathogenesis of brucellosis. Thirty-five goats were randomly assigned for experimental infection with B. melitensis strain 16M while open or during mid-gestation. Approximately half of the animals in each group were then treated with tulathromycin and subsequently assessed for the development of humoral responses to infection, clinical presentation, and bacterial dissemination and colonization. All animals, regardless of treatment group were successfully challenged with B. melitensis 16M demonstrated by bacterial recovery from conjunctival swabs and development of positive antibody titers. In goats infected while open, no animals aborted and Brucella was recovered from only one animal in tulathromycin-treated and one animal from the untreated group. Tulathromycin treatment of pregnant goats did not prevent abortion nor did it reduce bacterial dissemination, colonization, or shedding. Our data suggests that treatment of goats in mid-gestation with tulathromycin at the labeled dose does not influence disease pathogenesis or tissue colonization after experimental B. melitensis challenge.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

Carbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-D-mannopyranosyl-(1,2)-D-mannopyranoside), galactose (specifically carboxymethyl-β-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.

Biodegradable polyanhydride nanoparticles have been used as vaccine adjuvants and delivery vehicles. Upon parenteral administration, the surface characteristics of these nanoparticles may be modified by serum protein adsorption, likely affecting how the particles interact with antigen presenting cells. These studies investigated the role of the differential adsorption of serum proteins onto carbohydrate-functionalized and non-functionalized polyanhydride nanoparticles of different chemistries on interactions with dendritic cells (DCs). Di-mannose (a carboxymethyl-modified disaccharide of 1,2-α-linked-d-mannopyranoside) or glycolic acid was covalently linked to nanoparticles synthesized using copolymers of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH. Serum protein adsorption to CPH:SA nanoparticles enhanced particle internalization, but did not enhance uptake of CPTEG:CPH nanoparticles. When DCs were cultured with nanoparticles, the surface expression of MHC II, CD86, and CD206 was enhanced and mediated by both polymer chemistry and functionalization. Cytokine secretion by DCs was differentially modulated by both polymer chemistry and functionalization. The amount and profile of serum proteins adsorbed indicated that polymer hydrophobicity and functionalization enhanced protein adsorption. Overall polymer chemistry, functionalization, and serum protein adsorption influenced nanoparticle interactions with DCs and elucidating the complex relationships between these parameters and dendritic cell maturation will enable rational design of targeted vaccines and immunotherapies.

Polyanhydride nanoparticle-based vaccines (or nanovaccines) stabilize protein antigens, provide sustained antigen release leading to prolonged antigen presence, enhance activation of antigen presenting cells, and elicit protective immunity against respiratory infections upon challenge. However, induction of cell-mediated immunity when mice are immunized with polyanhydride nanovaccines has not been evaluated. Using a transgenic ovalbumin-specific T cell adoptive transfer model, we report the induction of antigen-specific cytotoxic CD8+ T cells expressing an effector memory phenotype by seven days after immunization with nanovaccine formulations. Furthermore, mice immunized with polyanhydride nanovaccines demonstrated enhanced recall responses after antigen re-exposure 35 days post-immunization indicating the activation and recruitment of antigen-specific memory CD8+ T cells to the site of antigen deposition.

Acute respiratory infections represent a significant portion of global morbidity and mortality annually. There is a critical need for efficacious vaccines against respiratory pathogens. To vaccinate against respiratory disease, pulmonary delivery is an attractive route because it mimics the route of natural infection and can confer both mucosal and systemic immunity. We have previously demonstrated that a single dose, intranasal vaccine based on polyanhydride nanoparticles elicited a protective immune response against Yersinia pestis for at least 40 weeks after immunization with F1-V. Herein, we investigate the effect of nanoparticle chemistry and its attributes on the kinetics and maturation of the antigen-specific serum antibody response. We demonstrate that manipulation of polyanhydride nanoparticle chemistry facilitated differential kinetics of development of antibody titers, avidity, and epitope specificity. The results provide new insights into the underlying role(s) of nanoparticle chemistry in providing long-lived humoral immunity and aid in the rational design of nanovaccine formulations to induce long-lasting and mature antibody responses.

Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, whereas co-infection of C57BL/6 (B6) mice leads to non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of the macrophage-mediated killing of L. amazonensis in vitro and a less robust antibody response. The mechanism that leads to healing of these lesions is not completely known, although our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To understand more completely this disparate clinical outcome to the same infection, we analyzed the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There were more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells, and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks postinfection. Interleukin (IL)-21 production and IL-21 receptor expression in both mouse strains, however, were similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. These data support the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.