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

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

An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Serum IgG Immunoglobulin Levels are Associated with Reduced PCR Detection of Mycoplasma bovis in Naturally Infected American Bison (Bison bison).

Mycoplasma bovis (M. bovis) is an important pathogen of American bison (Bison bison), associated with high morbidity and mortality epizootics of respiratory and reproductive disease. Despite the significant negative impact on bison health, little is known about the kinetics of disease and the host immune response to infection. To address these questions, a cohort of bison calves was created and serially sampled 5 times, once every 2-3 mo, over a 12-mo period. At each sampling period nasal swab samples were collected and tested by PCR for the presence of M. bovis. Serum samples were also collected and assessed for M. bovis-specific antibodies using both a commercial and an in-house ELISA. Overall, 19/41 bison (46.3%) had positive PCR tests, and 31/41 (75.6%) were seropositive. Over the course of the study, the frequency of PCR-positive nasal swabs and the ELISA scores decreased, although serum samples remained positive for at least 6 mo following the final positive PCR test. Bison were grouped according to results from the in-house ELISA into high-responder (n=7), low-responder (n=5), and seronegative (n=7) groups. M. bovis-specific IgG antibody levels were significantly elevated in the high-responder group compared to the low-responder and seronegative groups. The differences were statistically significant for 3/5 sampling periods. A trend toward increased IgG2 levels was observed in the high-responder group. High total IgG responses correlated with a decline in positive PCR tests from nasal swabs. These data provide evidence that a strong humoral response is beneficial and is probably involved in the clearance of M. bovis from bison.

Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection.

Human respiratory syncytial virus (HRSV) is a leading cause of death in young children and there are no FDA approved vaccines. Bovine RSV (BRSV) is antigenically similar to HRSV, and the neonatal calf model is useful for evaluation of HRSV vaccines. Here, we determined the efficacy of a polyanhydride-based nanovaccine encapsulating the BRSV post-fusion F and G glycoproteins and CpG, delivered prime-boost via heterologous (intranasal/subcutaneous) or homologous (intranasal/intranasal) immunization in the calf model. We compared the performance of the nanovaccine regimens to a modified-live BRSV vaccine, and to non-vaccinated calves. Calves receiving nanovaccine via either prime-boost regimen exhibited clinical and virological protection compared to non-vaccinated calves. The heterologous nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA, and induced similar clinical, virological and pathological protection as the commercial modified-live vaccine. Principal component analysis identified BRSV-specific humoral and cellular responses as important correlates of protection. The BRSV-F/G CpG nanovaccine is a promising candidate vaccine to reduce RSV disease burden in humans and animals.

Identification of a DRB3*011:01-restricted CD4+ T cell response against bovine respiratory syncytial virus fusion protein.

Although Human Respiratory Syncytial Virus (HRSV) is a significant cause of severe respiratory disease with high morbidity and mortality in pediatric and elderly populations worldwide there is no licensed vaccine. Bovine Respiratory Syncytial Virus (BRSV) is a closely related orthopneumovirus with similar genome structure and high homology between structural and nonstructural proteins. Like HRSV in children, BRSV is highly prevalent in dairy and beef calves and known to be involved in the etiology of bovine respiratory disease, in addition to being considered an excellent model for HRSV. Commercial vaccines are currently available for BRSV, though improvements in efficacy are needed. The aims of this study were to identify CD4+ T cell epitopes present in the fusion glycoprotein of BRSV, an immunogenic surface glycoprotein that mediates membrane fusion and a major target of neutralizing antibodies. Overlapping peptides representing three regions of the BRSV F protein were used to stimulate autologous CD4+ T cells in ELISpot assays. T cell activation was observed only in cells from cattle with the DRB3*011:01 allele by peptides from AA249-296 of the BRSV F protein. Antigen presentation studies with C-terminal truncated peptides further defined the minimum peptide recognized by the DRB3*011:01 allele. Computationally predicted peptides presented by artificial antigen presenting cells further confirmed the amino acid sequence of a DRB3*011:01 restricted class II epitope on the BRSV F protein. These studies are the first to identify the minimum peptide length of a BoLA-DRB3 class II-restricted epitope in BRSV F protein.

An intranasal recombinant NDV-BRSV Fopt vaccine is safe and reduces lesion severity in a colostrum-deprived calf model of RSV infection.

Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, yet no safe, effective vaccine is commercially available. Closely related bovine RSV (BRSV) causes respiratory disease in young calves, with many similar features to those seen in HRSV. We previously showed that a Newcastle disease virus (NDV)-vectored vaccine expressing the F glycoprotein of HRSV reduced viral loads in lungs of mice and cotton rats and protected from HRSV. However, clinical signs and pathogenesis of disease in laboratory animals following HRSV infection differs from that observed in human infants. Thus, we examined whether a similar vaccine would protect neonatal calves from BRSV infection. Codon-optimized rNDV vaccine (rNDV-BRSV Fopt) was constructed and administered to colostrum-deprived calves. The rNDV-BRSV Fopt vaccine was well-tolerated and there was no evidence of vaccine-enhanced disease in the upper airways or lungs of these calves compared to the non-vaccinated calves. We found two intranasal doses reduces severity of gross and microscopic lesions and decreases viral load in the lungs. Furthermore, serum neutralizing antibodies were generated in vaccinated calves. Finally, reduced lung CXC chemokine levels were observed in vaccinated calves after BRSV challenge. In summary, we have shown that rNDV-BRSV Fopt vaccine is safe in colostrum-deprived calves, and is effective in reducing lung lesions, and decreasing viral load in upper respiratory tract and lungs after challenge.

Comparative analysis of the specificity of monoclonal antibodies developed against the bottlenose dolphin, Tursiops truncatus, TNF-α, IL1-ß, IL-6, IL-8, IL-10 with monoclonal antibodies made against ovine IFN-γ bovine IL-17A and IL-1ß revealed they recognize epitopes conserved on dolphin and bovine orthologues.

Opportunities to include Cetancodontamorpha in the study of the evolution of the immune system in the clades of Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have increased with the use of the bottlenose dolphin, Tursiops truncatus, as a sentinel species to study the effects of environmental pollutants on the health of marine mammals. Efforts are currently underway to increase the number reagents needed for detailed studies. Thus far, screening of monoclonal antibodies (mAbs) made to leukocyte differentiation molecules (LDM) and the major histocompatibility (MHC) class I and class II molecules in Ruminantiamorpha have yielded some mAbs that recognize conserved epitopes expressed on orthologues in the bottlenose dolphin. More direct approaches are in progress to identify additional mAbs to bottlenose LDM and cytokines. As reported here, both direct and indirect approaches were used to identify mAbs specific for cytokines useful in monitoring the effects of environmental pollutants on the immune system. Immunization of mice with expressed bottlenose dolphin cytokines yielded mAbs specific for IFN-γ, TNF-α, IL-6, IL-8, IL-10, and IL-17A. Screening of previously developed mAbs used in livestock immunology research revealed mAbs developed against ovine IFN-γ and bovine IL-17 and IL-1ß recognize conserved epitopes in bottlenose dolphin orthologues. The mAbs identified in the present study expand the reagents available to study the function of the immune system in bottlenose dolphins and cattle.

Ex vivo activated CD4+ T cells from young calves exhibit Th2-biased effector function with distinct metabolic reprogramming compared to adult cows.

As maternal passive immunity wanes at 6-8 weeks, young calves must rely on their own naïve and developing immune system for protection against pathogens. Typically, an infection in the young induces a T cell-mediated response, which skews towards a Th2 phenotype and results in a reduced effector response. Our study examines the implications this transitional period of immunocompetency has on cellular metabolism in young calves, focusing on effector function of CD4+ T cells in comparison to those from adult cows. Results from sorted CD4+ T cells from young calves and adult cows activated by α-CD3:α-CD28, show that young calves exhibit a significantly greater propensity to produce the Th2 cytokine, IL-4, in comparison to IFN-γ. Concomitantly, cells from young calves and adult cows exhibit no statistical difference in cell surface marker expression induced by α-CD3:α-CD28 stimulation. Metabolically, activated CD4+ T cells from young calves show significantly greater utilization of mitochondrial respiration, measured by oxygen consumption rate (OCR), and greater glycolytic reserve, measured by extracellular acidification rate (ECAR). However, adult cows have a significantly higher change in glycolytic rate after α-CD3:α-CD28 stimulation compared to young calves. Further, CD4+ T cells from young calves have an increased mRNA expression signature associated with glycolytic metabolism (GAPDH, HK2, FBP1, HIF1A) and Th2-associated metabolic signaling (RPTOR) in comparison to adult cows. The distinct metabolic phenotype and associated gene expression in activated CD4+ T cells may be intrinsic drivers of the Th2-biased response by young calves. Additionally, CD4+ recent thymic emigrant cells (RTEs) may further contribute to altered effector function, as they are preferential precursors to Tregs, and based on the microenvironment, have the propensity to polarize toward Th2. Evaluation of T cell master transcription regulators, as well as measuring signal joint T cell receptor excision circles between young calves and adult cows, we observed a significantly increased proportion of RTEs from sorted CD4+ T cells. In this study, we show a unique metabolic profile exhibited by activated CD4+ T cells from young calves in which mitochondrial respiration and glycolytic capacity is significantly increased compared to adult cows.

Applications of Nanovaccines for Disease Prevention in Cattle.

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.

IMMUNOMEDIATOR GENE TRANSCRIPTION PROFILING IN BELUGA WHALE (DELPHINAPTERUS LEUCAS) CLINICAL CASES.

There is an unmet need for specific diagnostics of immune perturbations and inflammation in beluga whale (Delphinapterus leucas) clinical care. Quantitative real-time polymerase chain reaction (qPCR) has been used to measure immunomediator gene transcription in beluga whales. The study hypothesis was that a qPCR-based immunomediator assay would supplement routine clinical data with specific and sensitive information on immune status. Two beluga whale clinical cases provided an opportunity to test this hypothesis: a whale with a skin laceration and a whale with gastrointestinal inflammation. Mitogen-stimulated immunomediator gene transcription (MSIGT) was compared between the cases and healthy contact whales. In both case studies, mitogens increased transcription of IL1B, PTGS2 (Cox-2), TNF, HIF1A, and IL2 but decreased IL10 transcription in peripheral blood mononuclear cells (PBMC) from the abnormal whale over the control. Correlations were identified between most immunomediators tested and one or more standard blood clinical values. Considering all 15 immunomediators tested, the whale with gastrointestinal inflammation had a more unique MSIGT signature than the whale with a laceration. These results support further elucidation of beluga whale PBMC cytokine profiles for use as immune biomarkers.

The Immunology of Bovine Respiratory Disease: Recent Advancements.

Bovine respiratory disease (BRD) remains a leading cause of morbidity, mortality, and economic loss to the cattle industry. The continued high prevalence of the disease underlines a gap in understanding of the host immune response to respiratory infection. The host immune response is beneficial and detrimental, required for clearing the disease but often leading to tissue damage and long-term defects in lung function. This article highlights advancements made in understanding innate and adaptive immunity in BRD, factors that predispose animals to BRD, and novel intervention strategies that may lead to changes in the approach to treating and controlling BRD.

Lactation stage impacts the glycolytic function of bovine CD4+ T cells during ex vivo activation.

Dairy cattle undergo dynamic physiological changes over the course of a full lactation into the dry period, which impacts their immunocompetence. During activation, T cells undergo a characteristic rewiring to increase the uptake of glucose and metabolically reprogram to favor aerobic glycolysis over oxidative phosphorylation. To date it remains to be completely elucidated how the altered energetic demands associated with lactation in dairy cows impacts T cell metabolic reprogramming. Thus, in our ex vivo studies we have examined the influence of stage of lactation (early lactation into the dry period) on cellular metabolism in activated bovine CD4+ T cells. Results showed higher rates of glycolytic function in activated CD4+ T cells from late lactation and dry cows compared to cells from early and mid-lactation cows. Similarly, protein and mRNA expression of cytokines were higher in CD4+ T cells from dry cows than CD4+ T cells from lactating cows. The data suggest CD4+ T cells from lactating cows have an altered metabolic responsiveness that could impact the immunocompetence of these animals, particularly those in early lactation, and increase their susceptibility to infection.

Oxidative stress pathway gene transcription after bovine respiratory syncytial virus infection in vitro and ex vivo.

Studies in mouse and lamb models indicate important roles of reactive oxygen species (ROS) in the pathology and immune response to respiratory syncytial virus (RSV). The role of ROS in bovine RSV (BRSV) infection of calves remains unclear. BRSV naturally infects calves, leading to similar disease course, micro- and macro-lesions, and symptomology as is observed in RSV infection of human neonates. Furthermore, humans, lambs, and calves, but not mice, have an active lung oxidative system involving lactoperoxidase (LPO) and the dual oxidases (DUOX) 1 and 2. To gain insight into the role of ROS in the BRSV-infected lung, we examined gene expression in infected bovine cells using qPCR. A panel of 19 primers was used to assay ex vivo and in vitro BRSV-infected cells. The panel targeted genes involved in both production and regulation of ROS. BRSV infection significantly increased transcription of five genes in bovine respiratory tract cells in vitro and ex vivo. PTGS2 expression more than doubled in both sample types. Four transcripts varied significantly in lung lesions, but not non-lesion samples, compared with uninfected lung. This is the first report of the transcriptional profile of ROS-related genes in the airway after BRSV infection in the natural host.

Vitamin A deficiency impairs the immune response to intranasal vaccination and RSV infection in neonatal calves.

Respiratory syncytial virus (RSV) infection is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Vitamin A deficiency (VAD) is one of the most prevalent nutrition-related health problems in the world and is a significant risk factor in the development of severe respiratory infections in infants and young children. Bovine RSV (BRSV) is a primary cause of lower respiratory tract disease in young cattle. The calf model of BRSV infection is useful to understand the immune response to human RSV infection. We have previously developed an amphiphilic polyanhydride nanoparticle (NP)-based vaccine (i.e., nanovaccine) encapsulating the fusion and attachment proteins from BRSV (BRSV-NP). Calves receiving a single, intranasal dose of the BRSV-NP vaccine are partially protected from BRSV challenge. Here, we evaluated the impact of VAD on the immune response to the BRSV-NP vaccine and subsequent challenge with BRSV. Our results show that VAD calves are unable to respond to the mucosal BRSV-NP vaccine, are afforded no protection from BRSV challenge and have significant abnormalities in the inflammatory response in the infected lung. We further show that acute BRSV infection negatively impacts serum and liver retinol, rendering even well-nourished individuals susceptible to VAD. Our results support the use of the calf model for elucidating the impact of nutritional status on mucosal immunity and respiratory viral infection in infants and underline the importance of VA in regulating immunity in the respiratory mucosa.

Utility of the Neonatal Calf Model for Testing Vaccines and Intervention Strategies for Use against Human RSV Infection.

Respiratory syncytial virus (RSV) is a significant cause of pediatric respiratory tract infections. It is estimated that two-thirds of infants are infected with RSV during the first year of life and it is one of the leading causes of death in this age group worldwide. Similarly, bovine RSV is a primary viral pathogen in cases of pneumonia in young calves and plays a significant role in bovine respiratory disease complex. Importantly, naturally occurring infection of calves with bovine RSV shares many features in common with human RSV infection. Herein, we update our current understanding of RSV infection in cattle, with particular focus on similarities between the calf and human infection, and the recent reports in which the neonatal calf has been employed for the development and testing of vaccines and therapeutics which may be applied to hRSV infection in humans.

Relative virulence in bison and cattle of bison-associated genotypes of Mycoplasma bovis.

Mycoplasma bovis, a frequent contributor to polymicrobial respiratory disease in cattle, has recently emerged as a major health problem in North American bison. Strong circumstantial evidence suggests it can be the sole pathogen causing disease manifestations in outbreaks of mortality in bison, but direct evidence is lacking. The goal of this study was to compare clinical signs and lesions in bison and cattle experimentally infected with field isolates of M. bovis recovered from bison. Bison (n = 7) and cattle (n = 6), seronegative for anti-M. bovis IgG, were exposed intranasally to M. bovis and necropsied 4-6 weeks later. Blood and nasal swabs were collected on day 0 (before exposure), day 11 and at necropsy. Samples of lung, lymph node, liver and spleen were also collected at necropsy. The only clinical sign observed was an elevation in the core body temperature of bison during the first few weeks post-exposure. Grossly visible lesions were apparent at necropsy in the lungs of five bison and the lymph node of one bison, while none were evident in cattle. Histologic evaluation revealed moderate to severe pulmonary lesions in four bison but none in cattle. M. bovis was recovered from tissues demonstrating gross lesions and from the lymph nodes of one additional bison and two cattle. All animals seroconverted by the time of necropsy. These data provide the first direct evidence that M. bovis can be a sole or primary cause of respiratory disease in healthy bison, although the isolates used were unable to cause disease in healthy cattle.

Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf.

Human respiratory syncytial virus (HRSV) is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Bovine RSV (BRSV) is closely related to HRSV and a significant cause of morbidity in young cattle. BRSV infection in calves displays many similarities to RSV infection in humans, including similar age dependency and disease pathogenesis. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have shown promise as adjuvants and vaccine delivery vehicles due to their ability to promote enhanced immunogenicity through the route of administration, provide sustained antigen exposure, and induce both antibody- and cell-mediated immunity. Here, we developed a novel, mucosal nanovaccine that encapsulates the post-fusion F and G glycoproteins from BRSV into polyanhydride nanoparticles and determined the efficacy of the vaccine against RSV infection using a neonatal calf model. Calves receiving the BRSV-F/G nanovaccine exhibited reduced pathology in the lungs, reduced viral burden, and decreased virus shedding compared to unvaccinated control calves, which correlated with BRSV-specific immune responses in the respiratory tract and peripheral blood. Our results indicate that the BRSV-F/G nanovaccine is highly immunogenic and, with optimization, has the potential to significantly reduce the disease burden associated with RSV infection in both humans and animals.

Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus).

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 µg/mL ConA, while 1 µg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Genomic and Transcriptomic Analysis of Escherichia coli Strains Associated with Persistent and Transient Bovine Mastitis and the Role of Colanic Acid.

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. It is most often transient in nature, causing an infection that lasts 2 to 3 days. However, E. coli has been shown to cause a persistent infection in a minority of cases. Mechanisms that allow for a persistent E. coli infection are not fully understood. The goal of this work was to determine differences between E. coli strains originally isolated from dairy cattle with transient and persistent mastitis. Using RNA sequencing, we show gene expression differences in nearly 200 genes when bacteria from the two clinical phenotypes are compared. We sequenced the genomes of the E. coli strains and report genes unique to the two phenotypes. Differences in the wca operon, which encodes colanic acid, were identified by DNA as well as RNA sequencing and differentiated the two phenotypes. Previous work demonstrated that E. coli strains that cause persistent infections were more motile than those that cause transient infections. Deletion of genes in the wca operon from a persistent-infection strain resulted in a reduction of motility as measured in swimming and swarming assays. Furthermore, colanic acid has been shown to protect bacteria from complement-mediated killing. We show that transient-infection E. coli strains were more sensitive to complement-mediated killing. The deletion of genes from the wca operon caused a persistent-infection E. coli strain to become sensitive to complement-mediated killing. This work identifies important differences between E. coli strains that cause persistent and transient mammary infections in dairy cattle.

Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 µg/mL) and concanavalin A (1 µg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1ß, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.