Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Francisella tularensis novicida infection competence differs in cell lines derived from United States populations of Dermacentor andersoni and Ixodes scapularis.

In the United States, Dermacentor spp. are common vectors of Francisella tularensis subspecies (ssp.), while Ixodes scapularis is not, though the geographic distribution and host range of pathogen and tick overlap. To examine if differences in infection competence at the cellular level underpin these ecological differences, we evaluated the competence of D. andersoni (DAE100) and I. scapularis (ISE6) cell lines to support F. tularensis ssp. novicida (F. novicida) infection. Importantly, D. andersoni is a vector for both F. tularensis spp. tularensis, and F. novicida. We hypothesized F. novicida infection would be more productive in D. andersoni than in I. scapularis cells. Specifically, we determined if there are differences in F. novicida i) invasion, ii) replication, or iii) tick cell viability between DAE100 and ISE6 cells. We further examined the influence of temperature on infection kinetics. Both cell lines were permissive to F. novicida infection; however, there were significantly higher bacterial levels and mortality in DAE100 compared to ISE6 cells. Infection at environmental temperatures prolonged the time bacteria were maintained at high levels and reduced tick cell mortality in both cell lines. Identifying cellular determinants of vector competence is essential in understanding tick-borne disease ecology and designing effective intervention strategies.

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response.

In contrast to other species, α-Galactosylceramide (α-GalCer) is not an immunostimulatory NKT cell agonist in horses.

α-GalCer is a potent immunomodulatory molecule that is presented to NKT cells via the CD1 antigen-presenting system. We hypothesized that when used as an adjuvant α-GalCer would induce protective immune responses against Rhodococcus equi, an important pathogen of young horses. Here we demonstrate that the equine CD1d molecule shares most features found in CD1d from other species and has a suitable lipid-binding groove for presenting glycolipids to NKT cells. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi, and α-GalCer did not increase killing by CTL co-stimulated with R. equi antigen. Likewise, α-GalCer did not induce the lymphoproliferation of equine PBMC or increase the proliferation of R. equi-stimulated cells. Intradermal injection of α-GalCer in horses did not increase the recruitment of lymphocytes or cytokine production. Furthermore, α-GalCer-loaded CD1d tetramers, which have been shown to be broadly cross-reactive, did not bind equine lymphocytes. Altogether, our results demonstrate that in contrast to previously described species, horses are unable to respond to α-GalCer. This raises questions about the capabilities and function of NKT cells and other lipid-specific T lymphocytes in horses.

The equine CD1 gene family is the largest and most diverse yet identified.

The CD1 family is a group of non-polymorphic MHC class I-like molecules that present lipid-based antigens to T cells. Previous work in our laboratory demonstrated that cytotoxic T lymphocytes from immune adult horses recognize lipids from the cell wall of an important equine pathogen, Rhodococcus equi. These findings suggest an important role for the equine CD1 antigen presentation system in protective immune responses to microbial pathogens in the horse. In this study, we characterized and mapped the equine CD1 gene cluster. The equine genome was found to contain 13 complete CD1 genes; seven genes were classified as homologues of human CD1a, two CD1b, one CD1c, one CD1d, and two CD1e, making it the largest CD1 family to date. All but one of the eqCD1 molecules were expressed in all antigen-presenting cells investigated. The major amino acid differences between equine CD1 isoforms are located in the predicted antigen binding site, suggesting that a variety of lipid antigens can be presented. R. equi survives and replicates within professional phagocytes by arresting phagosome maturation between the early endosome and late phagosome. Based on the absence of a tyrosine sorting motif in all eqCD1a, CD1a molecules are predicted to co-localize with R. equi in the early endosome. Here, they could acquire lipid antigen and present it to T lymphocytes. The extraordinarily large number of CD1 molecules in the horse may reflect their crucial role in immunity to R. equi.

Early development of cytotoxic T lymphocytes in neonatal foals following oral inoculation with Rhodococcus equi.

Rhodococcus equi is an important respiratory pathogen of young foals for which a vaccine has long been sought. Two major impediments to effective vaccination are the functionally immature type I immune responses of neonatal foals and early exposure to the bacterium via the environment. Despite these obstacles, it appears that under specific circumstances foals can develop a protective immune response. In this study we investigated the protective mechanisms behind oral inoculation of foals with virulent R. equi bacteria. Two foals receiving an oral inoculum demonstrated accelerated development of R. equi specific cytotoxic T lymphocytes (CTL) as evidenced by significant lysis of R. equi infected, ELA-A mismatched cells at 3 weeks of age. As in a previous study, CTL were not detected until 5-6 weeks of age in two control foals. At each time point the ability of foal peripheral blood mononuclear cells (PBMC) to produce IFN-γ following stimulation with live R. equi or extracted cell wall lipids was similar to that of an adult horse control and between foals, regardless of treatment. These results provide a potential mechanism of protection which has previously been shown to occur following oral inoculation, and suggest that the early detection of CTL may be a useful marker for induction of protective immunity.

Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes.

Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. equi antigen with broadly reactive proteases did not significantly diminish the ability of the antigen to stimulate R. equi-specific CTLs. R. equi-specific CTLs were also shown to lyse target cells (equine macrophages) pulsed with an R. equi lipid extract. Analysis of the R. equi lipid by TLC and MS (MALDI-TOF and ES) indicated that the extracted antigen consisted of three primary fractions: trehalose monomycolate (TMM), trehalose dimycolate (TDM) and cardiolipin (CL). ELA-A-mismatched cells pulsed with purified TMM and CL, but not the TDM fraction, were recognized and lysed by R. equi-specific CTLs. Because of their role in immune clearance and pathogenesis, transcription of the cytokines gamma interferon (IFN-gamma) and interleukin-4 (IL-4) was also measured in response to R. equi lipids by using real-time PCR; elevated IFN-gamma, but not IL-4, was associated with host clearance of the bacteria. The whole-cell R. equi lipid and all three R. equi lipid fractions resulted in marked increases in IFN-gamma transcription, but no increase in IL-4 transcription. Together, these data support the hypothesis that immune recognition of unique lipids in the bacterial cell wall is an important component of the protective immune response to R. equi. The results also identify potential lipid antigens not previously shown to be recognized by CTLs in an important, naturally occurring actinomycete bacterial pathogen.

Serum antibodies against human albumin in critically ill and healthy dogs.

OBJECTIVE: To characterize the magnitude and duration of the antibody response against human albumin (HA) in critically ill and healthy dogs. DESIGN: Cohort and cross-sectional study. ANIMALS: Fourteen critically ill dogs that received 25% HA as part of their treatment protocol, 2 healthy dogs with no known previous exposure to HA that received 2 infusions of 25% HA (positive control dogs), and 47 healthy dogs and 21 critically ill dogs with no known exposure to HA (negative control dogs). PROCEDURES: An ELISA to detect IgG against HA was developed. Serum samples were obtained from the critically ill dogs prior to infusion of HA, at the time of hospital discharge, and 4 to 6 weeks and 6 months after HA administration. Serum samples were obtained at 2- to 4-week intervals from both positive control dogs for 101 weeks. A single serum sample was obtained from each of the negative control dogs. RESULTS: All 14 critically ill dogs developed serum IgG against HA. Peak antibody response was detected 4 to 6 weeks after HA administration. In both positive control dogs, IgG against HA was detected 10 days after HA administration and continued past 97 weeks. The peak antibody response was detected at 3 weeks in 1 dog and at 9 weeks in the other. Five of the 68 (7%) negative control dogs had a positive antibody response. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that dogs developed a pronounced IgG response following exposure to HA and that some dogs with no history of HA administration were positive for anti-HA IgG.

Adverse reactions suggestive of type III hypersensitivity in six healthy dogs given human albumin.

CASE DESCRIPTION: 6 healthy dogs given human albumin solution as part of a study were examined following development of an immediate hypersensitivity reaction (1 dog) and signs suggestive of a type III hypersensitivity reaction (all 6 dogs). CLINICAL FINDINGS: All 6 dogs were healthy prior to administration of human albumin solution. One dog developed signs of an immediate hypersensitivity reaction, characterized by vomiting and facial edema, during administration of human albumin solution. All 6 dogs developed signs of a delayed adverse reaction 5 to 13 days after administration of human albumin solution. Initial clinical signs included lethargy, lameness, edema, cutaneous lesions indicative of vasculitis, vomiting, and inappetance. TREATMENT AND OUTCOME: In the dog with signs of immediate hypersensitivity, signs resolved after administration of human albumin solution was discontinued and diphenhydramine was administered. Supportive treatment was provided after dogs developed signs of a delayed adverse reaction. Four dogs recovered, but 2 dogs died despite treatment. All 6 dogs were found to have antihuman albumin antibodies. There was no evidence of contamination of the human albumin solution. CLINICAL RELEVANCE: Findings suggest that administration of human albumin solution in healthy dogs with normal serum albumin concentrations may result in signs of a type III hypersensitivity reaction.

Rhodococcus equi secreted antigens are immunogenic and stimulate a type 1 recall response in the lungs of horses immune to R. equi infection.

Rhodococcus equi is an opportunistic pathogen in immunocompromised humans and an important primary pathogen in young horses. Although R. equi infection can produce life-threatening pyogranulomatous pneumonia, most foals develop a protective immune response that lasts throughout life. The antigen targets of this protective response are currently unknown; however, Mycobacterium tuberculosis is a closely related intracellular pathogen and provides a model system. Based on previous studies of M. tuberculosis protective antigens released into culture filtrate supernatant (CFS), a bacterial growth system was developed for obtaining R. equi CFS antigens. Potential immunogens for prevention of equine rhodococcal pneumonia were identified by using immunoblots. The 48-h CFS contained five virulence-associated protein bands that migrated between 12 and 24 kDa and were recognized by sera from R. equi-infected foals and immune adult horses. Notably, the CFS contained the previously characterized proteins VapC, VapD, and VapE, which are encoded by genes on the R. equi virulence plasmid. R. equi CFS was also examined for the ability to stimulate a type 1-like memory response in immune horses. Three adult horses were challenged with virulent R. equi, and cells from the bronchoalveolar lavage fluid were recovered before and 1 week after challenge. In vitro stimulation of pulmonary T-lymphocytes with R. equi CFS resulted in significant proliferation and a significant increase in gamma interferon mRNA expression 1 week after challenge. These results were consistent with a memory effector response in immune adult horses and provide evidence that R. equi CFS proteins are antigen targets in the immunoprotective response against R. equi infection.

Analysis of anamnestic immune responses in adult horses and priming in neonates induced by a DNA vaccine expressing the vapA gene of Rhodococcus equi.

Rhodococcus equi remains one of the most important pathogens of early life in horses, yet conventional vaccines to prevent rhodococcal pneumonia have not been successful. DNA vaccination offers an alternative to conventional vaccines with specific advantages for immunization of neonates. We developed a DNA vaccine expressing the vapA gene (pVR1055vapA) that induced an anamnestic response characterized by virulence associated protein A (VapA)-specific IgG antibodies in sera and bronchoalveolar lavage fluid (BALF) as well as VapA-specific proliferation of pulmonary lymphocytes when tested in adult ponies. In contrast, none of the adults receiving the control plasmid responded. To determine if pVR1055vapA induced VapA-specific responses in the foal, the targeted age group for vaccination against R. equi, 10 naïve foals were randomly assigned at birth to two groups of five. At 8-15 days of age (day 1), foals were vaccinated by intranasal and intradermal (i.d.) routes with either pVR1055vapA or the negative control pVR1055vapA_rev. All foals were DNA boosted at day 14 and protein boosted at day 30 with either recombinant VapA or recombinant CAT (control group). Prior to the protein boost, neither group developed VapA-specific immune responses. However, at day 45, two of the VR1055vapA-vaccinated foals had increased titers of VapA-specific IgGb, IgM and IgGa in the sera, and IgG in the BALF. The induction of the opsonizing isotypes IgGa and IgGb has been previously shown to be associated with protection against R. equi. No VapA-specific immune responses were detected in the control group. This study indicates that the DNA vaccine effectively stimulates anamnestic systemic and pulmonary immune responses in adult horses. The results in foals suggest that the DNA vaccine also primed a subset of immunized neonates. These data support further development and modification to produce a DNA vaccine to more effectively prime neonatal foals.

Identification of pulmonary T-lymphocyte and serum antibody isotype responses associated with protection against Rhodococcus equi.

Rhodococcus equi infects and causes pneumonia in foals between 2 and 4 months of age but does not induce disease in immunocompetent adults, which are immune and remain clinically normal upon challenge. Understanding the protective response against R. equi in adult horses is important in the development of vaccine strategies, since those mechanisms likely reflect the protective phenotype that an effective vaccine would generate in the foal. Twelve adult horses were challenged with virulent R. equi and shown to be protected against clinical disease. Stimulation of cells obtained from bronchoalveolar lavage fluid with either R. equi or the vaccine candidate protein VapA resulted in significant proliferation and a significant increase in the level of gamma interferon (IFN-gamma) expression by day 7 postchallenge. The levels of interleukin-4 expression were also increased at day 7 postchallenge; however, this increase was not antigen specific. Anamnestic increases in the levels of binding to R. equi and VapA of all immunoglobulin G (IgG) antibody isotypes [IgGa, IgGb, IgG(T)] examined were detected postchallenge. The levels of R. equi- and VapA-specific IgGa and IgGb antibodies, the IgG isotypes that preferentially opsonize and fix complement in horses, were dramatically enhanced postchallenge. The antigen-specific proliferation of bronchoalveolar lavage fluid cells, the levels of IFN-gamma expression by these cells, and the anamnestic increases in the levels of opsonizing IgG isotypes are consistent with stimulation of a memory response in immune adult horses and represent correlates for vaccine development in foals.