CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Characterization of the adaptive cellular and humoral immune responses to persistent colonization of Brucella abortus strain RB51 in a Jersey cow.

Brucella abortus strain RB51 is the commercial cattle vaccine used in the United States (US) and many parts of the world against bovine brucellosis. RB51 was licensed for use in 1996, and it has been shown to be safe and efficacious in cattle, eliciting humoral and cellular responses in calves and adult animals. In 2017, an epidemiological trace-back investigation performed by the Centers for Disease Control and Prevention (CDC) identified human cases of brucellosis caused by infection with RB51. These infections resulted from the consumption of unpasteurized dairy products, which were traced back to otherwise healthy animals that were shedding RB51 in their milk. At the current time, six adult Jersey cows have been identified in the U.S. that are shedding RB51 in milk. One of the RB51 shedding cattle was obtained and housed at the National Animal Disease Center (NADC) for further study. Improved understanding of host cellular and humoral immune responses to RB51 in persistently colonized cattle may be achieved by the characterization of responses in shedding animals. We hypothesized, based on the lack of RB51 clearance, that the RB51 shedder animal has a diminished adaptive cellular immune response to RB51. Our data demonstrate that in the presence of persistent RB51 infection, there is a lack of peripheral anti-RB51 CD4+ T cell responses and a concurrently high anti-RB51 IgG humoral response. By understanding the mechanisms that result in RB51 persistence, the development of improved interventions or vaccinations for brucellosis may be facilitated, which would provide public health benefits, including reducing the risks associated with the consumption of non-pasteurized milk products.

An injectable subunit vaccine containing Elongation Factor Tu and Heat Shock Protein 70 partially protects American bison from Mycoplasma bovis infection.

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.

Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

White-tailed deer (Odocoileus virginianus) have emerged as a reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence and infection across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk (Cervus elaphus canadensis) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk.

Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis).

BACKGROUND: Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. RESULTS: The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. CONCLUSIONS: The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

Wildlife Immune Responses to Mycobacterium bovis and to Bacille of Calmette-Guerin.

Bovine tuberculosis (bTB) is a zoonotic bacterial disease presenting public health, veterinary, and economic threats around the globe. Although cattle producers rely on regular testing and management practices to minimize domestic herd exposure, wildlife species around the world continue to be the main reservoirs for disease. Wildlife reservoirs for bTB include the Eurasian badger (Meles meles) in Great Britain and Ireland, the brushtail possum (Trichosurus vulpecula) in New Zealand, wild boar (Sus scrofa) in Spain, as well as white-tailed deer (Odocoileus virginianus) in the United States and red deer (Cervus elaphus) in Spain. Although all reservoir species share the ability to infect cattle, they differ in transmission capability, disease pathogenesis, diagnostic detection, and vaccination strategies. In this review, bTB interactions with these wildlife reservoirs are discussed, illustrating the need to address bTB disease in wildlife hosts to achieve eradication in domestic livestock.

Pulmonary hamartoma in an elk calf.

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.

Immune Responses and Efficacy of Brucella Abortus Strain RB51 in Bison After Delivery in a Dry Dart Formulation or by Parenteral Inoculation.

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.

Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo.

This study examined the humoral and cellular response of cattle vaccinated with two commercial leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4+ and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4+ and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic.IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.

Quantification of Brucella abortus population structure in a natural host.

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 108 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.

Lesion Material From Treponema-Associated Hoof Disease of Wild Elk Induces Disease Pathology in the Sheep Digital Dermatitis Model.

A hoof disease among wild elk (Cervus elaphus) in the western United States has been reported since 2008. Now present in Washington, Oregon, Idaho, and California, this hoof disease continues to spread among elk herds suggesting an infectious etiology. Causing severe lesions at the hoof-skin junction, lesions can penetrate the hoof-horn structure causing severe lameness, misshapen hooves, and in some cases, sloughed hooves leaving the elk prone to infection, malnutrition, and premature death. Isolated to the feet, this disease has been termed treponeme-associated hoof disease due to the numerous Treponema spp. found within lesions. In addition to the Treponema spp., treponeme-associated hoof disease shares many similarities with digital dermatitis of cattle and livestock including association with several groups of anaerobic bacteria such as Bacteroides, Clostridia, and Fusobacterium, neutrophilic inflammatory infiltrate, and restriction of the disease to the foot and hoof tissues. To determine if there was a transmissible infectious component to this disease syndrome, elk lesion homogenate was used in a sheep model of digital dermatitis. Ten animals were inoculated with lesion material and lesion development was followed over 7 weeks. Most inoculated feet developed moderate to severe lesions at 2- or 4-weeks post-inoculation timepoints, with 16 of 18 feet at 4 weeks also had spirochetes associated within the lesions. Histopathology demonstrated spirochetes at the invading edge of the lesions along with other hallmarks of elk hoof disease, neutrophilic inflammatory infiltrates, and keratinocyte erosion. Treponema-specific PCR demonstrated three phylotypes associated with elk hoof disease and digital dermatitis were present. Serum of infected sheep had increased anti-Treponema IgG when compared to negative control sheep and pre-exposure samples. Analysis of the bacterial microbiome by sequencing of the bacterial 16S rRNA gene showed a community structure in sheep lesions that was highly similar to the elk lesion homogenate used as inoculum. Bacteroidies, Fusobacterium, and Clostridia were among the bacterial taxa overrepresented in infected samples as compared to negative control samples. In conclusion, there is a highly transmissible, infectious bacterial component to elk treponeme-associated hoof disease which includes several species of Treponema as well as other bacteria previously associated with digital dermatitis.

Influence of species of negative control sera on results of a brucellosis fluorescence polarization assay.

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.

Loci Associated With Antibody Response in Feral Swine (Sus scrofa) Infected With Brucella suis.

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 including Brucella 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 with B. suis using 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 for Brucella spp. 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 to B. suis infection, 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 to B. suis infection and to more effectively translate estimates of Brucella spp. antibody prevalence among feral swine to disease control management action.

Case Report: Fading Elk Syndrome in a Herd of Captive Elk (Cervus elaphus) in the North American Midwest.

Fading elk syndrome, or chronic ill-thrift of elk, is a disease associated with abomasal parasitism with Ostertagia species, of which elk appear to be particularly susceptible. While this syndrome has been extensively reported to affect wapiti-type red deer hybrids farmed in New Zealand since the mid 1980's, there is only a single report of this disease in North America. Here, we report a case of fading elk syndrome in a herd of 34 elk (Cervus elaphus) in Ames, Iowa, at the National Animal Disease Center. Analysis of complete blood counts were unremarkable, but blood chemistry demonstrated a severe hypoalbuminemia. Fecal floatations were also unremarkable, and non-diagnostic. Histological examination of tissues collected at necropsy revealed proliferative abomasitis and nematodes consistent with Ostertagia spp. Anthelmintic treatment consisting of a combination of pour-on Cydectin® and injectable Noromectin Plus®, at double the recommended dose for cattle, showed positive results, as all remaining animals in the herd recovered. The work presented here is the first report of naturally-acquired disease in a herd of captive elk used for research and sheds light on this seldomly-reported disease in North America.

Enhancing the Detection of Brucella-Specific CD4+ T Cell Responses in Cattle via in vitro Antigenic Expansion and Restimulation.

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.

Bovine immune response to leptospira antigen in different novel adjuvants and vaccine delivery platforms.

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.

Characterization of the NLRP1 inflammasome response in bovine species.

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.

Tulathromycin treatment does not affect bacterial dissemination or clearance of Brucella melitensis 16M following experimental infection of goats.

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.

Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis.

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.

Effect of a DNA-based immunostimulant on growth, performance, and expression of inflammatory and immune mediators in beef calves abruptly weaned and introduced to a complete ration.

The effect of a DNA immunostimulant on inflammatory and immune responses, performance, and health in calves following abrupt weaning and introduction to a concentrate diet was tested. Sixty-four single source Angus crossbred steers were weaned on day 1 and assigned to receive a DNA immunostimulant (TRT) or saline (CON) on days 0, 2, 4, and 6. On day 0, steers received clostridial and respiratory vaccines and anthelmintic; they were then transported 2 h, allocated to pens (n = 8 per pen), and introduced to total mixed ration. Daily intake, ADG, and feed efficiency were measured. Serum haptoglobin, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) were assayed by ELISA or AlphaLISA on days 0, 2, 4, 6, 14, and 28; serum-neutralizing antibodies (SNA) to bovine herpesvirus-1 and bovine viral diarrhea virus-1 (BVDV-1) were quantified on days 0, 28, 68, and 135. In a subset of cattle (n = 6 to 8 per treatment group), the percent macrophages and activated gamma delta (γδ) T cells in blood was determined by flow cytometry on days 2 and 6, and expression of mRNA for TNF-α, interferon-gamma (IFN-γ), IL-4, and IL-10 by stimulated blood mononuclear cells was assessed by real-time reverse transcriptase PCR on day 6. After 70 d, cattle were shipped 1,205 km to a feedlot and performance and health were followed. There was a significant effect of time on serum TNF-α, IL-1ß, haptoglobin, and SNA (P < 0.001); the range in concentration among cattle on each day was large. The ratio of IFN-γ to IL-4 expression was significantly higher (P = 0.03) for TRT cattle, suggesting that treatment activated T-helper type 1 cells. There was a trend toward an improved feed conversion (P = 0.10) for TRT steers over the 70-d backgrounding period. There was no effect of treatment on feedlot performance or carcass merit (P > 0.10). During backgrounding, 1 TRT steer died of enterocolitis. In spite of backgrounding, cattle experienced an outbreak of bovine respiratory disease (BRD) in the feedlot and 1 of 31 TRT cattle and 5 of 32 CON cattle died of BRD. The immunostimulant modified some immune responses during backgrounding. Large variability in inflammatory responses during backgrounding indicated that events around weaning induce systemic inflammation that varies substantially among cattle.