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1.  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-γ) 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-γ 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.
doi:10.1128/CDLI.9.6.1270-1276.2002
PMCID: PMC130094  PMID: 12414760
2.  Immunoglobulin G Subisotype Responses of Pneumonic and Healthy, Exposed Foals and Adult Horses to Rhodococcus equi Virulence-Associated Proteins 
Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised humans. Replication of virulent isolates within macrophages correlates with the presence of a large plasmid which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH), whose functions are unknown. Although cell-mediated immunity is thought to be crucial in eliminating R. equi infection, antibody partially protects foals. The antibody response to both VapA and VapC was similar in six adult horses and six naturally exposed but healthy foals, as well as in eight foals with R. equi pneumonia. The immunoglobulin G (IgG) subisotype response of pneumonic foals to Vap proteins was significantly IgGb biased and also had a trend toward higher IgGT association compared to the isotype association of antibody in adult horses and healthy exposed foals. This suggests that in horses, IgGb and IgGT are Th2 isotypes and IgGa is a Th1 isotype. Furthermore, it suggests that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response. Pneumonic foals had significantly more antibody to VapD and VapE than did healthy exposed foals. This may indicate a difference in the expression of these two Vap proteins during persistent infection. Alternatively, in pneumonic foals the deviation of the immune response toward VapD and VapE may reflect a bias unfavorable to R. equi resistance. These data indicate possible age-related differences in the equine immune response affecting Th1-Th2 bias as well as antibody specificity bias, which together favor the susceptibility of foals to R. equi pneumonia.
doi:10.1128/CDLI.10.3.345-351.2003
PMCID: PMC154967  PMID: 12738629
3.  Experimental Infection of Neonatal Foals with Rhodococcus equi Triggers Adult-Like Gamma Interferon Induction▿  
Rhodococcus equi is a facultative intracellular pathogen that causes pneumonia in young foals but does not induce disease in immunocompetent adult horses. Clearance of R. equi depends mainly on gamma interferon (IFN-γ) production by T lymphocytes, whereas the predominance of interleukin 4 (IL-4) is detrimental. Young foals, like neonates of many other species, are generally deficient in the ability to produce IFN-γ. The objective of this study was to compare the cytokine profiles, as well as cell-mediated and antibody responses, of young foals to those of adult horses following intrabronchial challenge with R. equi. The lymphoproliferative responses of bronchial lymph node (BLN) cells to concanavalin A were significantly higher in foals than in adult horses. In contrast, adult horses had significantly higher lymphoproliferative responses to R. equi antigens than did foals. Infected foals had significantly lower IL-4 mRNA expression but significantly higher IFN-γ expression and IFN-γ/IL-4 ratio in R. equi-stimulated BLN lymphocytes than did infected adults. Infection with R. equi in foals resulted in a significant increase in the percentage of T lymphocytes and CD4+ T lymphocytes in bronchoalveolar lavage fluid in association with a significant decrease in the percentage of these cell populations in BLNs. Infection of foals also resulted in a marked increase in serum immunoglobulin Ga (IgGa) and IgGb levels, resulting in concentrations in serum that were significantly higher than those of adult horses. This study demonstrates that the immune response to R. equi in foals is not biased toward IL-4 and is characterized by the predominant induction of IFN-γ.
doi:10.1128/CVI.00042-07
PMCID: PMC1951072  PMID: 17409222
4.  Expression of Equi Merozoite Antigen 2 during Development of Babesia equi in the Midgut and Salivary Gland of the Vector Tick Boophilus microplus 
Journal of Clinical Microbiology  2003;41(12):5803-5809.
Equi merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equi proteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileria spp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equi transitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equi stages in the midgut lumen and midgut epithelial cells of Boophilus microplus nymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equi in salivary glands of adult Boophilus microplus. In addition, quantification of B. equi in the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equi organisms in the midgut. In contrast, there was no conclusive evidence that B. equi EMA-1 was expressed in either the Boophilus microplus midgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equi EMA-2 in Boophilus microplus provides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesia proteins for testing transmission-blocking immunity.
doi:10.1128/JCM.41.12.5803-5809.2003
PMCID: PMC308990  PMID: 14662988
5.  Lymphocytes and Macrophages Are Infected by Theileria equi, but T Cells and B Cells Are Not Required to Establish Infection In Vivo 
PLoS ONE  2013;8(10):e76996.
Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.
doi:10.1371/journal.pone.0076996
PMCID: PMC3792048  PMID: 24116194
6.  Specific immune responses are required to control parasitemia in Babesia equi infection. 
Infection and Immunity  1994;62(5):1909-1913.
Horses possessing a normal immune system and spleen often control infection caused by Babesia equi. However, splenectomized horses are unable to control B. equi infection and usually succumb to the infection. To investigate the role of the spleen in the control of B. equi infection in the absence of specific immune responses, two 1-month-old foals with severe combined immunodeficiency (SCID) and two age-matched normal foals were inoculated with B. equi. The SCID foals became febrile seven days postinoculation and developed terminal parasitemias of 41 and 29%. The SCID foals had greater than 50% decreases in indices of total erythrocytes, packed-cell volumes, and hemoglobin concentrations. Both SCID foals were euthanized in extremis at 10 days postinoculation. As expected, the serum of the SCID foals lacked detectable antibodies to B. equi antigens. In contrast, the normal foals inoculated with B. equi produced detectable anti-erythrocyte-stage parasite antibodies by 7 days and controlled clinical disease by 12 days postinoculation. Although SCID foals lack functional T and B lymphocytes, they do possess complement, macrophages, granulocytes, and natural killer cells, as well as a spleen. Therefore, the data indicate that specific immune responses are required to control B. equi parasitemia but are not required for erythrocyte lysis in infected horses. Furthermore, the spleen is not able to control B. equi parasitemia in the absence of specific immune responses to parasite antigens.
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PMCID: PMC186438  PMID: 8168957
7.  Ability of the Vector Tick Boophilus microplus To Acquire and Transmit Babesia equi following Feeding on Chronically Infected Horses with Low-Level Parasitemia 
Journal of Clinical Microbiology  2005;43(8):3755-3759.
The protozoan parasite Babesia equi replicates within erythrocytes. During the acute phase of infection, B. equi can reach high levels of parasitemia, resulting in a hemolytic crisis. Horses that recover from the acute phase of the disease remain chronically infected. Subsequent transmission is dependent upon the ability of vector ticks to acquire B. equi and, following development and replication, establishment of B. equi in the salivary glands. Although restriction of the movement of chronically infected horses with B. equi is based on the presumption that ticks can acquire and transmit the parasite at low levels of long-term infection, parasitemia levels during the chronic phase of infection have never been quantified, nor has transmission been demonstrated. To address these epidemiologically significant questions, we established long-term B. equi infections (>1 year), measured parasitemia levels over time, and tested whether nymphal Boophilus microplus ticks could acquire and, after molting to the adult stage, transmit B. equi to naive horses. B. equi levels during the chronic phase of infection ranged from 103.3 to 106.0/ml of blood, with fluctuation over time within individual horses. B. microplus ticks fed on chronically infected horses with mean parasite levels of 105.5 ± 100.48/ml of blood acquired B. equi, with detection of B. equi in the salivary glands of 7 to 50% of fed ticks, a range encompassing the percentage of positive ticks that had been identically fed on a horse in the acute phase of infection with high parasitemia levels. Ticks that acquired B. equi from chronically infected horses, as well as those fed during the acute phase of infection, successfully transmitted the parasite to naive horses. The results unequivocally demonstrated that chronically infected horses with low-level parasitemia are competent mammalian reservoirs for tick transmission of B. equi.
doi:10.1128/JCM.43.8.3755-3759.2005
PMCID: PMC1233951  PMID: 16081906
8.  Equine Neonates Have Attenuated Humoral and Cell-Mediated Immune Responses to a Killed Adjuvanted Vaccine Compared to Adult Horses ▿  
Clinical and Vaccine Immunology : CVI  2010;17(12):1896-1902.
The objectives of this study were to compare relative vaccine-specific serum immunoglobulin concentrations, vaccine-specific lymphoproliferative responses, and cytokine profiles of proliferating lymphocytes between 3-day-old foals, 3-month-old foals, and adult horses after vaccination with a killed adjuvanted vaccine. Horses were vaccinated intramuscularly twice at 3-week intervals with a vaccine containing antigens from bovine viral respiratory pathogens to avoid interference from maternal antibody. Both groups of foals and adult horses responded to the vaccine with a significant increase in vaccine-specific IgGa and IgG(T) concentrations. In contrast, only adult horses and 3-month-old foals mounted significant vaccine-specific total IgG, IgGb, and IgM responses. Vaccine-specific concentrations of IgM and IgG(T) were significantly different between all groups, with the highest concentrations occurring in adult horses, followed by 3-month-old foals and, finally, 3-day-old foals. Only the adult horses mounted significant vaccine-specific lymphoproliferative responses. Baseline gamma interferon (IFN-γ) and interleukin-4 (IL-4) concentrations were significantly lower in 3-day-old foals than in adult horses. Vaccination resulted in a significant decrease in IFN-γ concentrations in adult horses and a significant decrease in IL-4 concentrations in 3-day-old foals. After vaccination, the ratio of IFN-γ/IL-4 in both groups of foals was significantly higher than that in adult horses. The results of this study indicate that the humoral and lymphoproliferative immune responses to this killed adjuvanted vaccine are modest in newborn foals. Although immune responses improve with age, 3-month-old foals do not respond with the same magnitude as adult horses.
doi:10.1128/CVI.00328-10
PMCID: PMC3008191  PMID: 20943883
9.  Getting to Grips with Strangles: An Effective Multi-Component Recombinant Vaccine for the Protection of Horses from Streptococcus equi Infection 
PLoS Pathogens  2009;5(9):e1000584.
Streptococcus equi subspecies equi (S. equi) is a clonal, equine host-adapted pathogen of global importance that causes a suppurative lymphodendopathy of the head and neck, more commonly known as Strangles. The disease is highly prevalent, can be severe and is highly contagious. Antibiotic treatment is usually ineffective. Live attenuated vaccine strains of S. equi have shown adverse reactions and they suffer from a short duration of immunity. Thus, a safe and effective vaccine against S. equi is highly desirable. The bacterium shows only limited genetic diversity and an effective vaccine could confer broad protection to horses throughout the world. Welsh mountain ponies (n = 7) vaccinated with a combination of seven recombinant S. equi proteins were significantly protected from experimental infection by S. equi, resembling the spontaneous disease. Vaccinated horses had significantly reduced incidence of lymph node swelling (p = 0.0013) lymph node abscessation (p = 0.00001), fewer days of pyrexia (p = 0.0001), reduced pathology scoring (p = 0.005) and lower bacterial recovery from lymph nodes (p = 0.004) when compared with non-vaccinated horses (n = 7). Six of 7 vaccinated horses were protected whereas all 7 non-vaccinated became infected. The protective antigens consisted of five surface localized proteins and two IgG endopeptidases. A second vaccination trial (n = 7+7), in which the IgG endopeptidases were omitted, demonstrated only partial protection against S. equi, highlighting an important role for these vaccine components in establishing a protective immune response. S. equi shares >80% sequence identity with Streptococcus pyogenes. Several of the components utilized here have counterparts in S. pyogenes, suggesting that our findings have broader implications for the prevention of infection with this important human pathogen. This is one of only a few demonstrations of protection from streptococcal infection conferred by a recombinant multi-component subunit vaccine in a natural host.
Author Summary
Numerous research groups have vaccinated, using recombinant antigens, against streptococcal infections in mouse model systems and shown protection. We have here demonstrated efficient protective vaccination of the natural host, the horse, using recombinant antigens. Streptococcus equi subspecies equi (S. equi) is an equine host-adapted and highly contagious pathogen of global importance. Six out of seven Welsh mountain ponies vaccinated with a combination of seven recombinant S. equi proteins were protected from experimental infection as assessed by clinical examination, pyrexia, lymph node swelling, inflammation, bacterial recovery, and post mortem examination. The protective antigens consisted of five surface localized proteins and two endopeptidases that are specific for IgG; the latter were shown to be of major importance for efficacy. Several of the antigens used here have similarities in Streptococcus pyogenes, implying that our findings are of importance for development of a vaccine against this important human pathogen.
doi:10.1371/journal.ppat.1000584
PMCID: PMC2736577  PMID: 19763180
10.  High-Level Expression and Purification of a Truncated Merozoite Antigen-2 of Babesia equi in Escherichia coli and Its Potential for Immunodiagnosis 
Journal of Clinical Microbiology  2003;41(3):1147-1151.
The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.
doi:10.1128/JCM.41.3.1147-1151.2003
PMCID: PMC150322  PMID: 12624044
11.  Identification of immunoglobulin heavy-chain isotypes of specific antibodies of horse 46 group B meningococcal antiserum. 
Journal of Clinical Microbiology  1982;15(2):324-329.
Hyperimmune horse serum from a single animal (horse 46) immunized with group B (strain B-11) meningococcal vaccine provides a standardized, readily available diagnostic reagent used in primary isolation medium and for serogrouping of meningococci. Identification of the heavy-chain isotypes of specific anticapsular polysaccharide and anti-lipopolysaccharide isolated from horse 46 serum revealed a differential distribution in the occurrence of immunoglobulin classes. Meningococcal anticapsular antibodies of horse 46 serum were restricted predominately to the immunoglobulin M (IgM) class, with only trace amounts of IgGa present, whereas anti-lipopolysaccharide concomitantly produced showed a heterogeneity in its heavy-chain isotypes, consisting of IgM, IgGa, IgGb, moderate amounts of IgB, and a small amount of IgA.
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PMCID: PMC272085  PMID: 6802867
12.  Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila, and human granulocytic Ehrlichia. 
Journal of Clinical Microbiology  1995;33(5):1098-1103.
Homology in the 16S rDNAs shows that the agent of human granulocytic ehrlichiosis (HGE) is closely related to the veterinary pathogens Erlichia equi and Erlichia phagocytophila. After HGE, patients develop antibodies reactive with E. equi and E. phagocytophila; thus, we hypothesized that these species are closely related and share significant antigenicity. Antisera from humans, horses, dogs, and cattle were tested by indirect fluorescent-antibody assay (IFA) for antibodies reactive with E. equi and other ehrlichiae and tested by immunoblot to identify the specific reactions with E. equi. All convalescent-phase sera from human patients with HGE and from animals infected or immunized with E. equi or E. phagocytophila had antibodies reactive with E. equi by IFA; no reactions with Ehrlichia chaffeensis occurred with these sera, and only one horse naturally infected with E. equi had a serologic reaction against Ehrlichia sennetsu. Human and animal sera obtained after infection or immunization with other Ehrlichia, Rickettsia, and Bartonella species did not react with E. equi by IFA. E. equi immunoblots revealed as many as 19 bands with equine anti-E. equi serum. All HGE agent, E. equi, and E. phagocytophila antisera tested reacted with a 44-kDa antigen of E. equi, while other anti-Ehrlichia spp. sera reacted with this antigen rarely or not at all. HGE agent, E. equi, and E. phagocytophila antisera but not other sera also reacted occasionally with 25-, 42-, and 100-kDa antigens. Most sera reacted with antigens between approximately 56 and 75 kDa, probably heat shock proteins. The HGE agent, E. equi, and E. phagocytophila share significant antigenicity by IFA and immunoblot.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC228112  PMID: 7542262
13.  Immunoglobulin and specific antibody responses to Rhodococcus (Corynebacterium) equi infection in foals as measured by enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1986;23(5):943-947.
Humoral immune response to intestinal Rhodococcus (Corynebacterium) equi in horses was studied by enzyme-linked immunosorbent assay. Anti-R. equi immunoglobulin M (IgM), IgG, and IgA antibodies were demonstrated in the healthy horse population. Adult horse levels of anti-R. equi IgM and IgG antibodies were reached by 5 to 9 weeks of age in two healthy newborn foals. R. equi was recovered from the foals in the range of 10(3) to 10(4) per g of intestinal contents. A 1-week-old foal was infected with R. equi by mouth daily for 9 weeks. The foal did not show any clinical signs of illness. Anti-R. equi IgM antibody values in the foal increased about 5 to 8 weeks after initial inoculation, similar to the naturally occurring immune response to intestinal R. equi. There were differences among the antibody responses to R. equi in healthy horses, foals with suspected infection, and infected foals. These results suggest that exposure to R. equi is widespread in the horse population and that intestinal R. equi is the most important source of antigenic stimulation for a naturally occurring immune response in horses.
PMCID: PMC268755  PMID: 3711280
14.  Comparative genomic analysis and phylogenetic position of Theileria equi 
BMC Genomics  2012;13:603.
Background
Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites.
Results
The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp.
Conclusions
The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.
doi:10.1186/1471-2164-13-603
PMCID: PMC3505731  PMID: 23137308
Apicomplexa; Parasite; Vaccine; Horse; Vector-borne disease
15.  Recombinant Streptococcus equi Proteins Protect Mice in Challenge Experiments and Induce Immune Response in Horses  
Infection and Immunity  2004;72(6):3228-3236.
Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (α2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.
doi:10.1128/IAI.72.6.3228-3236.2004
PMCID: PMC415648  PMID: 15155624
16.  A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites. 
Infection and Immunity  1991;59(7):2412-2417.
Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses.
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PMCID: PMC258026  PMID: 1711016
17.  Persistently Infected Horses Are Reservoirs for Intrastadial Tick-Borne Transmission of the Apicomplexan Parasite Babesia equi▿  
Infection and Immunity  2008;76(8):3525-3529.
Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥104 organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.
doi:10.1128/IAI.00251-08
PMCID: PMC2493223  PMID: 18490466
18.  Immunochromatographic Test for Simultaneous Serodiagnosis of Babesia caballi and B. equi Infections in Horses 
Clinical and Vaccine Immunology  2006;13(5):553-555.
An immunochromatographic test for the simultaneous detection of Babesia caballi- and B. equi-specific antibodies (BceICT) was developed using a recombinant B. caballi 48-kDa rhoptry protein (rBc48) and a recombinant truncated B. equi merozoite antigen 2 (rEMA-2t). An evaluation of the ability of the BceICT to detect antibodies in sera from uninfected horses and experimentally infected horses showed high sensitivities and specificities of 83.3% (10/12 sera) and 92.9% (52/56 sera), respectively, for the anti-B. caballi antibody and 94.1% (16/17 sera) and 88.2% (45/51 sera), respectively, for the anti-B. equi antibody. Results from the detection of antibodies in field-collected sera indicated that the BceICT results corresponded with those of enzyme-linked immunosorbent assays (ELISA), showing 91.8% correspondence (67/73 sera) for B. caballi and 95.9% correspondence (70/73 sera) for B. equi, and that the BceICT results also corresponded with the ICT for B. caballi and for B. equi, both of which were 98.2% (55/56 sera). The comparable results of the ICT and ELISA and the simplicity and rapidity of the performance of the ICT suggest that the BceICT would be a feasible test for the simultaneous serodiagnosis of both agents of equine babesiosis in the field.
doi:10.1128/CVI.13.5.553-555.2006
PMCID: PMC1459648  PMID: 16682475
19.  Mucosal nasopharyngeal immune responses of horses to protein antigens of Streptococcus equi. 
Infection and Immunity  1985;47(3):623-628.
Mucosal nasopharyngeal immunoglobulin A (IgA) and IgG responses to proteins of Streptococcus equi were studied in horses after the experimental production of strangles. S. equi-specific IgA and IgG titers in nasopharyngeal mucus were much higher in samples from animals 1 to 2 weeks after challenge than in samples from control animals. Although IgA was the major immunoglobulin in nasal mucus, there was more antibody activity associated with IgG as measured by radioimmunoassay. Great differences between the specificities of antibodies in nasal mucus and in serum were detected. IgA and IgG of mucus origin recognized only two major proteins with molecular weights of about 41,000 and 46,000 in acid extracts of S. equi and gave no detectable reaction with culture supernatant proteins. Only one protein of about 62,000 molecular weight was recognized in acid extracts of an equine strain of S. zooepidemicus. In contrast, immunoglobulins in serum recognized a great variety of proteins in culture supernatants and acid extracts of S. equi and S. zooepidemicus which did not include those proteins recognized by immunoglobulins in mucus. These findings provide good evidence for the independence of the local and systemic immune responses of the horse to S. equi. Horses rechallenged shortly after recovery from the first infection were resistant to challenge with an inoculum of S. equi 10 times greater than that to which they were originally susceptible. This resistance appeared to be independent of the levels of bactericidal antibody in serum. We therefore suggest that immunity to S. equi infection is mediated by locally produced nasopharyngeal antibodies.
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PMCID: PMC261338  PMID: 3972443
20.  Cloning of a Novel Babesia equi Gene Encoding a 158-Kilodalton Protein Useful for Serological Diagnosis 
In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.
doi:10.1128/CDLI.12.2.334-338.2005
PMCID: PMC549306  PMID: 15699430
21.  Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi. 
Journal of Clinical Microbiology  1992;30(12):3122-3126.
Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.
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PMCID: PMC270599  PMID: 1280648
22.  Monoclonal Antibody against Babesia equi: Characterization and Potential Application of Antigen for Serodiagnosis 
Journal of Clinical Microbiology  1998;36(7):1835-1839.
Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.
PMCID: PMC104937  PMID: 9650921
23.  Detection of Antibodies to Babesia equi in Horses by a Latex Agglutination Test Using Recombinant EMA-1 
A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.
doi:10.1128/CDLI.8.3.645-646.2001
PMCID: PMC96117  PMID: 11329474
24.  Protective Effects of Passively Transferred Merozoite-Specific Antibodies against Theileria equi in Horses with Severe Combined Immunodeficiency 
Theileria equi immune plasma was infused into young horses (foals) with severe combined immunodeficiency. Although all foals became infected following intravenous challenge with homologous T. equi merozoite stabilate, delayed time to peak parasitemia occurred. Protective effects were associated with a predominance of passively transferred merozoite-specific IgG3.
doi:10.1128/CVI.05301-11
PMCID: PMC3255952  PMID: 22038847
25.  Rhodococcus equi-Infected Macrophages Are Recognized and Killed by CD8+ T Lymphocytes in a Major Histocompatibility Complex Class I-Unrestricted Fashion  
Infection and Immunity  2004;72(12):7073-7083.
The goal of this research was to examine the role of cytotoxic T lymphocytes (CTL) in the control of Rhodococcus equi and specifically to determine if R. equi-specific CD8+ CTL occurred in the blood of immune horses. Equine peripheral blood mononuclear cells stimulated with antigen-presenting cells either infected with R. equi or exposed to soluble R. equi antigen lysed R. equi-infected target cells. Lysis was decreased to background by depletion of either CD2+ or CD3+ cells, indicating that the effector cell had a T-lymphocyte, but not NK cell, phenotype. Stimulation induced an increased percentage of CD8+ T cells in the effector population, and depletion of CD8+ T cells resulted in significantly decreased lysis of infected targets. Killing of R. equi-infected macrophages by effector cells was equally effective against autologous and equine leukocyte antigen A (classical major histocompatibility complex [MHC] class I) mismatched targets. To evaluate potential target antigens, target cells were infected with either virulent (80.6-kb plasmid-containing) or avirulent (plasmid-cured) R. equi. The degree of lysis was not altered by the presence of the plasmid, providing evidence that the virulence plasmid, which is required for survival within macrophages, was not necessary for recognition and killing of R. equi-infected cells. These data indicate that immunocompetent adult horses develop R. equi-specific CD8+ CTL, which may play a role in immunity to R. equi. The apparent lack of restriction via classical MHC class I molecules suggests a novel or nonclassical method of antigen processing and presentation, such as presentation by CD1 or other nonclassical MHC molecules.
doi:10.1128/IAI.72.12.7073-7083.2004
PMCID: PMC529141  PMID: 15557631

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