Comparison of the tyrosine aminotransferase cDNA and genomic DNA sequences of normal mink and mink affected with tyrosinemia type II.

Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.

Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.

Immune reconstitution prevents continuous equine infectious anemia virus replication in an Arabian foal with severe combined immunodeficiency: lessons for control of lentiviruses.

Acute infection with equine infectious anemia virus (EIAV), a lentivirus of horses, results in a persistent high-level viremia in Arabian foals affected with severe combined immunodeficiency (SCID). This observation argues against the idea that the transient nature of acute lentiviral viremia is solely a function of viral population dynamics. To extend these studies, EIAV-specific immune reconstitution was attempted prior to EIAV challenge in two SCID foals, using adoptively transferred virus-stimulated lymphocytes derived from persistently EIAV-infected half sibling donors. Following transfer, lymphocyte engraftment occurred in one foal, and EIAV-specific cytotoxic T lymphocytes as well as neutralizing antibody activity developed. Following a brief period of plasma viremia in this foal, EIAV replication was controlled and plasma virus could not be detected by RT-PCR or culture. These results provide further direct evidence that a specific immune response is required for termination of plasma viremia in acute lentiviral infections.

A subset of Cowdria ruminantium genes important for immune recognition and protection.

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.

CD4(+) T lymphocytes from calves immunized with Anaplasma marginale major surface protein 1 (MSP1), a heteromeric complex of MSP1a and MSP1b, preferentially recognize the MSP1a carboxyl terminus that is conserved among strains.

Native major surface protein 1 (MSP1) of the ehrlichial pathogen Anaplasma marginale induces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4(+) T-lymphocyte responses have not been evaluated. CD4(+) T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-gamma), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginale and related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4(+) T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4(+) T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains of A. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-gamma production by CD4(+) T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4(+) T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.

A simpler, more sensitive competitive inhibition enzyme-linked immunosorbent assay for detection of antibody to malignant catarrhal fever viruses.

An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2-3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV.

Long-term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti-CD4 monoclonal antibody treatment.

In vivo depletion of lymphocyte subsets is a direct approach used for dissection of the mechanisms of protective immunity. Long-term in vivo depletion of bovine T lymphocyte subpopulations with monoclonal antibody (mAb) treatment alone has been difficult to achieve. The objective of this study was to determine whether both thymectomy and anti-CD4 mAb treatment would optimize long-term in vivo depletion of functional bovine CD4+ T lymphocytes. Calves were thymectomized and treated with high doses of anti-CD4 mAb (approximately 5 mg/kg) over 4 days followed by subsequent lower doses (approximately 0.3 mg/kg) administered twice weekly for an additional 7 weeks. Depletion of CD4+ T lymphocytes from blood, spleen and peripheral lymph nodes was significantly improved in thymectomized calves compared to thymus-intact anti-CD4 mAb-treated calves. Significant differences in percentages of CD4+ T lymphocytes between thymectomized and thymus-intact calves were sustained for the duration of the 8-week study. Depletion of CD4+ T lymphocytes from thymectomized calves resulted in complete abrogation of lymphoproliferative responses to ovalbumin. In addition, thymectomized calves treated with anti-CD4 mAb had significantly reduced immunoglobulin G1 and no detectable immunoglobulin G2 ovalbumin-specific antibody responses compared to thymus-intact anti-CD4 mAb-treated calves. The results of this study demonstrate that both thymectomy and treatment with anti-CD4 mAb are required for long-term in vivo depletion of functional bovine CD4+ T lymphocytes.

Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens.

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp2 and msp3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.

Highly conserved regions of the immunodominant major surface protein 2 of the genogroup II ehrlichial pathogen Anaplasma marginale are rich in naturally derived CD4+ T lymphocyte epitopes that elicit strong recall responses.

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4(+) T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-gamma, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.

Monoclonal antibody binding to a surface-exposed epitope on Cowdria ruminantium that is conserved among eight strains.

Monoclonal antibodies (MAb) binding to Cowdria ruminantium elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with Ehrlichia canis antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development.

Monoclonal antibody differentiation of Mycoplasma mycoides subsp. mycoides small-colony strains causing contagious bovine pleuropneumonia from less important large-colony strains.

Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine Mycoplasma mycoides subsp. mycoides small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for M. mycoides subsp. mycoides small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.

Expression of polymorphic msp1beta genes during acute anaplasma Marginale rickettsemia.

Immunization of cattle with native MSP1 induces protection against Anaplasma marginale. The native immunogen is composed of a single MSP1a protein and multiple, undefined MSP1b polypeptides. In addition to the originally sequenced gene, designated msp1beta(F1), we identified three complete msp1beta genes in the Florida strain: msp1beta(F2), msp1beta(F3), and msp1beta(F4). Each of these polymorphic genes encodes a structurally unique MSP1b protein, and unique transcripts can be identified during acute A. marginale rickettsemia. The structural polymorphism is clustered in discrete variable regions, and each MSP1b protein results from a unique mosaic of five variable regions. Although each of the MSP1b proteins in the Florida strain contains epitopes recognized by serum antibody induced by protective immunization with the native MSP1 complex, the variable regions also include epitopes expressed by some but not all of the MSP1b proteins. These data support testing recombinant vaccines composed of the multiple antigenically and structurally unique MSP1b proteins combined with MSP1a in order to mimic the efficacy of native MSP1 immunization.

Crossreactive neutralizing antibodies induced by immunization with caprine arthritis-encephalitis virus surface glycoprotein.

Four Saanen goats were immunized with affinity purified gp135 surface glycoprotein (SU) of caprine arthritis-encephalitis virus isolate 79-63 (CAEV-63) and evaluated for homologous and crossreactive serum neutralizing antibodies. CAEV-63 neutralizing antibodies were detected in all goats after seven immunizations with SU in Quil A adjuvant. Sera from three goats neutralized an independent CAEV isolate (CAEV-Co). However, serum from one goat did not detectably neutralize heterologous CAEV-Co and inhibited CAEV-Co neutralization by another serum.

An in vivo model to investigate lymphocyte-mediated immunity during acute hemoparasitic infections. Use of a monoclonal antibody to selectively deplete CD4+ T lymphocytes from thymectomized calves.

Thymectomized calves were selectively depleted of CD4+ T lymphocytes with a monoclonal antibody (mAb) specific for the bovine CD4 monomer (ILA-11). Calves were treated with high loading doses of ILA-11 during the first week of the study then treated with subsequent lower maintenance doses. Depletion of CD4+ T lymphocytes was assessed weekly by flow cytometric analysis of PBMC and mononuclear cells from lymph node and spleen biopsies. Treatment with high doses of ILA-11 resulted in rapid and marked depletion of CD4+ T lymphocytes from the peripheral blood, peripheral lymph nodes, and spleen. Although CD4+ T lymphocytes slowly returned to the peripheral blood, peripheral lymph nodes, and spleen by day 21 post-treatment, the numbers of CD4+ T lymphocytes in depleted calves remained below pre-depletion levels for the duration of the study. CD4+ T lymphocytes failed to be effectively depleted from a non-thymectomized calf treated with the mAb ILA-11. Development of a T lymphocyte depletion model in thymectomized calves will permit testing of the hypothesis that CD4+ T lymphocytes and IFN-gamma are required in cattle for control of acute anaplasmosis. In subsequent planned studies, thymectomized calves depleted of CD4+ T lymphocytes will be experimentally infected with A. marginale and parameters of disease compared between depleted and non-depleted calves.

Interleukin-12 as an adjuvant promotes immunoglobulin G and type 1 cytokine recall responses to major surface protein 2 of the ehrlichial pathogen Anaplasma marginale.

Anaplasma marginale is a tick-transmitted pathogen of cattle closely related to the human ehrlichiae, Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (HGE). These pathogens have in common a structurally conserved outer membrane protein (OMP) designated the major surface protein 2 (MSP-2) in A. marginale and HGE and OMP-1 in E. chaffeensis. Protective immunity against ehrlichial pathogens is believed to require induction of gamma interferon (IFN-gamma) and opsonizing immunoglobulin (Ig) subclasses directed against OMP epitopes that, in concert, activate macrophages for phagocytosis and killing. Because interleukin-12 (IL-12) acts as an adjuvant for protein immunization to induce IFN-gamma and protective immunity against intracellular pathogens, we hypothesized that as an adjuvant with MSP-2, IL-12 would augment type 1 recall responses to A. marginale. IL-12 was coadsorbed with MSP-2 to alum and shown to significantly enhance IFN-gamma production by lymph node cells (LNC) and LNC-derived CD4(+) T-cell lines from immunized calves following recall stimulation with A. marginale. LNC proliferation and IL-2 production were also enhanced in IL-12-treated calves. Elevated recall proliferative responses by peripheral blood mononuclear cells were still evident 9 months after immunization. Serum IgG levels were consistently increased in IL-12 immunized calves, predominantly due to higher IgG1 responses. The results support the use of IL-12 coadsorbed with OMP of ehrlichial pathogens in alum to amplify both antibody and type-1 cytokine responses important for protective immunity.

Natural variation of equine infectious anemia virus Gag protein cytotoxic T lymphocyte epitopes.

Two defined cytotoxic T lymphocyte (CTL) epitopes from equine infectious anemia virus (EIAV)-infected horses, equine leukocyte alloantigen (ELA)-A5.1-restricted epitope 18a, and ELA-A9-restricted epitope 28b-1 were evaluated for conservation among three wild-type EIAV strains. Epitope 18a variation occurred in all three wild-type EIAV strains, while epitope 28b-1 varied in one strain. Further, 12% amino acid changes occurred in the Gag proteins of a recently isolated wild-type strain, documenting a much greater Gag protein variation than previously reported. Evaluation of epitope 18a among two virus isolates from sequential disease episodes in a single horse, H513 (ELA-A5.1/A8), demonstrated that no variation that affected CTL recognition occurred. H513 PBMC had CTLm to epitope 18a before the occurrence of disease episodes caused by viruses expressing epitope 18a; however, the frequencies were low (5-15/10(6) PBMC). Later in infection there was an absence of disease episodes associated with an increase in CTLm frequency to EIAV(WSU5)-infected targets, but not epitope 18a-pulsed targets. Therefore, if CTLm to EIAV epitopes were involved in maintaining the carrier state in H513, they recognized epitopes other than 18a.

Biased immunoglobulin G1 isotype responses induced in cattle with DNA expressing msp1a of Anaplasma marginale.

Immunization with the native major surface protein 1 (MSP1) (a heterodimer containing disulfide and noncovalently bonded polypeptides designated MSP1a and MSP1b) of the erythrocytic stage of Anaplasma marginale conferred protection against homologous challenge (G. H. Palmer, A. F. Barbet, W. C. Davis, and T. C. McGuire, Science 231:1299-1302, 1986). The MSP1a polypeptide possesses a conserved neutralization-sensitive epitope. In the present study, the immune response to DNA-mediated immunization using msp1a was studied. The plasmid pVCL/MSP1a, which encodes the complete msp1a gene of A. marginale under the control of human cytomegalovirus immediate-early enhancer/promoter and intron A, was constructed. The immune responses elicited by immunization with pVCL/MSP1a into cardiotoxin-induced regenerating muscle were evaluated in mice and cattle. Antibody reactive with native MSP1a was detected in pooled sera of immunized BALB/c mice 3 weeks following primary immunization. Two calves seronegative for A. marginale were immunized four times, at weeks 0, 3, 7, and 13, with pVCL/MSP1a. By 8 weeks, both calves responded to MSP1a with an antibody titer of 1:100, which peaked at 1:1,600 and 1:800 by 16 weeks after the initial immunization. Interestingly, immunoblotting with anti-immunoglobulin G1 (anti-IgG1) and anti-IgG2 specific monoclonal antibodies revealed a restricted IgG1 anti-MSP1a response in both animals. T-lymphocyte lines, established after the fourth immunization, proliferated specifically against A. marginale homogenate and purified MSP1 in a dose-dependent manner. These data provide a basis for an immunization strategy to direct bovine immune responses by using DNA vaccine vectors containing single or multiple genes encoding major surface proteins of A. marginale.

Neutralizing antibody responses and evolution of antigenic variants in monozygotic twin lambs infected with phenotypically distinct ovine lentiviruses.

Ovine lentivirus (OvLV) isolates 85/34 (OvLV 34) and 84/28 (OvLV 28) were initially characterized as phenotypically distinct "rapid/high" and "slow/low" strains based on replication kinetics, syncytiogenesis, and cell lysis in vitro. In the present study, sera from OvLV-34- or OvLV-28-infected monozygotic twin lambs defined these virus strains as distinct neutralization serotypes. We also show that immune recognition of at least one OvLV neutralization epitope is influenced by genetic differences between lambs. Additional studies determined the neutralization phenotype of virus isolates from alveolar macrophages of OvLV-34- or OvLV-28-infected lambs, evaluated the role of neutralizing antibodies in selection and persistence of antigenic variants, and related the severity of OvLV-induced lymphoid interstitial pneumonia (LIP) to the evolution of neutralization variants. These studies demonstrate that (i) macrophage-associated OvLV neutralization variants can arise in the presence or the absence of neutralizing antibodies directed to inoculum viruses, (ii) OvLV variants persist in macrophages in the presence of serum neutralizing antibodies, and (iii) the emergence of OvLV variants is apparently unrelated to the severity of LIP.