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

Disseminated \u3ci\u3eLeishmania infantum\u3c/i\u3e infection in two sibling foxhounds due to possible vertical transmission

Two sibling foxhounds born to a Leishmania seropositive bitch were presented after testing seropositive for Leishmania. Leishmania infantum infection was detected via histopathology, culture, and quantitative polymerase chain reaction (q-PCR). This is the first report of natural infection with Leishmania infantum with the possibility for vertical transmission in North America. Infection disséminée à Leishmania infantum chez deux chiots Fox hound d’une même portée reliée possiblement à une transmission verticale. Deux chiots Fox hound d’une même portée nés d’une mère séropositive à Leishmania ont été présentés après un contrôle sérologique positif. Une infection à Leishmania infantum a été détectée par histopathologie, culture et amplification en chaîne par polymérase quantitative (ACP-q). Il s’agit du premier rapport d’infection naturelle par Leishmania infantum possiblement relié à une transmission verticale en Amérique du Nord

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

A pseudomolecule assembly of the Rocky Mountain elk genome

Rocky Mountain elk (Cervus canadensis) populations have significant economic implications to the cattle industry, as they are a major reservoir for Brucella abortus in the Greater Yellowstone area. 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 Hi-C 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 eight putative gene losses in elk

Fecal and vaginal microbiota of vaccinated and non-vaccinated pregnant elk challenged with Brucella abortus

IntroductionBrucella abortus is the causative agent of brucellosis in cattle and in humans, resulting in economic losses in the agricultural sector and representing a major threat to public health. Elk populations in the American Northwest are reservoirs for this bacterium and transmit the agent to domestic cattle herds. One potential strategy to mitigate the transmission of brucellosis by elk is vaccination of elk populations against B. abortus; however, elk appear to be immunologically distinct from cattle in their responses to current vaccination strategies. The differences in host response to B. abortus between cattle and elk could be attributed to differences between the cattle and elk innate and adaptive immune responses. Because species-specific interactions between the host microbiome and the immune system are also known to affect immunity, we sought to investigate interactions between the elk microbiome and B. abortus infection and vaccination.MethodsWe analyzed the fecal and vaginal microbial communities of B. abortus-vaccinated and unvaccinated elk which were challenged with B. abortus during the periparturient period.ResultsWe observed that the elk fecal and vaginal microbiota are similar to those of other ruminants, and these microbial communities were affected both by time of sampling and by vaccination status. Notably, we observed that taxa representing ruminant reproductive tract pathogens tended to increase in abundance in the elk vaginal microbiome following parturition. Furthermore, many of these taxa differed significantly in abundance depending on vaccination status, indicating that vaccination against B. abortus affects the elk vaginal microbiota with potential implications for animal reproductive health.DiscussionThis study is the first to analyze the vaginal microbiota of any species of the genus Cervus and is also the first to assess the effects of B. abortus vaccination and challenge on the vaginal microbiome

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

Transplacental Transmission of Leishmania infantum as a Means for Continued Disease Incidence in North America

Dogs are a favored feeding source for sand flies that transmit human L. infantum infection. Zoonotic visceral leishmaniasis (ZVL) is an emerging problem in some U.S. dog breeds, with over 20% of at-risk Foxhounds infected. Although classically Leishmania is transmitted by infected sand flies which exist in the United States, no role has yet been determined for vector-borne transmission. Means of ongoing L. infantum transmission in U.S. dogs is unknown. Possibilities include transplacental and horizontal/venereal transmission. Aims for this study were to establish whether transplacental transmission occurred in Leishmania-infected U.S. dogs and determine the effect of this transmission on immune recognition of Leishmania. This novel report describes wide-spread infection as identified by kqPCR in 8 day-old pups born to a naturally-infected, seropositive U.S. dog with no travel history. This is the first report of transplacental transmission of L. infantum in naturally-infected dogs in North America. Evidence that mom-to-pup transmission of ZVL may continue disease in an otherwise non-endemic region has significant implications on current control strategies for ZVL. Determining frequency of vertical transmission and incorporating canine sterilization with vector control may have a more significant impact on ZVL transmission to people in endemic areas than current control efforts

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa)

Pseudorabies virus (PRV)—the causative agent of Aujeszky’s disease—was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk–based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome–wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA–SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA–SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production

Leishmaniasis: immunologic indicators of clinical progression and mechanisms of immune modulation

Leishmaniasis is a group of vector-borne diseases caused by obligate intracellular protozoan parasites of the genus Leishmania. In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. In order to differentiate infection vs. progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in Foxhounds within the United States. Several key changes in immunological parameters should be considered to differentiate infection versus clinical disease, including a dramatic rise in IgG production, loss of antigen-specific PBMC proliferation, and IFN-gamma production, concomitant IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and immuno-dysregulation mirrors that observed in human patients indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 or other therapies. The murine model of cutaneous leishmaniasis using L. amazonensis infection provides an excellent model to study host-Leishmania interactions that may lead to a non-productive immune response. Dendritic cells (DC) are professional antigen presenting cells, critical for induction and regulation of T cell immune responses against pathogens. Initiation of productive immune responses against Leishmania depends on the successful transition of DC from an immature to a mature phenotype, characterized by high CD40 surface expression and IL-12 production. This productive immune response is frequently seen in response to L. major infection. Characterization of draining lymph node CD11c+ DC from L. amazonensis or L. major promastigote-infected mice revealed that by 7 days post-infection, CD11c+ cells from the draining lymph node of L. amazonensis promastigote-infected mice have significantly reduced CD40 surface expression, and there was a significant decrease in the number of IL-12p40-producing cells. Analysis of MAP kinase signaling revealed that infection of DC in vitro with L. amazonensis resulted in increased ERK phosphorylation. Inhibition of ERK in vitro or in vivo led to enhanced DC surface CD40 expression and in vitro increased IL-12p40 production. Peak ERK1/2 phosphorylation was observed 4 hours post-infection in vitro, when parasites are primarily present within intracellular phagolysosome-based parasitophorous vacuoles. Intracellular activation of ERK1/2 from phagolysosomes requires the recruitment and association of ERK1/2-specific scaffolding proteins, including p14/MP1 and MORG1, on the surface of this intracellular compartment. Here we identify scaffolding module co-localization, specifically the endosomal ERK-related proteins MP1 and MORG1 with L. amazonensis-containing, LAMP2 positive-phagolysosomes in DC. This is the first time pathogen ERK1/2 activation is correlated to intracellular-induced ERK activation and identifies novel signaling cascade components which interact with the L. amazonensis parasitophorous vacuole.</p

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