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Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.

Strain superinfection occurs when a second pathogen strain infects a host already carrying a primary strain. Anaplasma marginale superinfection occurs when the second strain carries a variant repertoire different from that of the primary strain, and the epidemiologic consequences depend on the relative efficiencies of tick-borne transmission of the two strains. Following strain superinfection in the reservoir host, we tested whether the presence of two A. marginale (sensu lato) strains that differed in transmission efficiency altered the transmission phenotypes in comparison to those for single-strain infections. Dermacentor andersoni ticks were fed on animals superinfected with the Anaplasma marginale subsp. centrale vaccine strain (low transmission efficiency) and the A. marginale St. Maries strain (high transmission efficiency). Within ticks that acquired both strains, the St. Maries strain had a competitive advantage and replicated to significantly higher levels than the vaccine strain. The St. Maries strain was subsequently transmitted to naïve hosts by ticks previously fed either on superinfected animals or on animals singly infected with the St. Maries strain, consistent with the predicted transmission phenotype of this strain and the lack of interference due to the presence of a competing low-efficiency strain. The vaccine strain was not transmitted by either singly infected or coinfected ticks, consistent with the predicted transmission phenotype and the lack of enhancement due to the presence of a high-efficiency strain. These results support the idea that the strain predominance in regions of endemicity is mediated by the intrinsic transmission efficiency of specific strains regardless of occurrence of superinfection.

Background: With the recent completion of numerous sequenced bacterial genomes, notable advances have been made in understanding the level of conservation between various species. However, relatively little is known about the genomic diversity among strains. We determined the complete genome sequence of the Florida strain of Anaplasma marginale, and near complete (>96%) sequences for an additional three strains, for comparative analysis with the previously fully sequenced St. Maries strain genome., Results: These comparisons revealed that A. marginale has a closed-core genome with few highly plastic regions, which include the msp2 and msp3 genes, as well as the aaap locus. Comparison of the Florida and St. Maries genome sequences found that SNPs comprise 0.8% of the longer Florida genome, with 33.5% of the total SNPs between all five strains present in at least two strains and 3.0% of SNPs present in all strains except Florida. Comparison of genomes from three strains of Mycobacterium tuberculosis, Bacillus anthracis, and Nessieria meningiditis, as well as four Chlamydophila pneumoniae strains found that 98.8%-100% of SNPs are unique to each strain, suggesting A. marginale, with 76.0%, has an intermediate level of strain-specific SNPs. Comparison of genomes from other organisms revealed variation in diversity that did not segregate with the environmental niche the bacterium occupies, ranging from 0.00% to 8.00% of the larger pairwise-compared genome., Conclusion: Analysis of multiple A. marginale strains suggests intracellular bacteria have more variable SNP retention rates than previously reported, and may have closed-core genomes in response to the host organism environment and/or reductive evolution.

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EMPhi strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMPhi strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.

A new pathogen strain can penetrate an immune host population only if it can escape immunity generated against the original strain. This model is best understood with influenza viruses, in which genetic drift creates antigenically distinct strains that can spread through host populations despite the presence of immunity against previous strains. Whether this selection model for new strains applies to complex pathogens responsible for endemic persistent infections, such as anaplasmosis, relapsing fever, and sleeping sickness, remains untested. These complex pathogens undergo rapid within-host antigenic variation by using sets of chromosomally encoded variants. Consequently, immunity is developed against a large repertoire of variants, dramatically changing the scope of genetic change needed for a new strain to evade existing immunity and establish coexisting infection, termed strain superinfection. Here, we show that the diversity in the alleles encoding antigenic variants between strains of a highly antigenically variant pathogen was equal to the diversity within strains, reflecting equivalent selection for variants to overcome immunity at the host population level as within an individual host. This diversity among strains resulted in expression of nonoverlapping variants that allowed a new strain to evade immunity and establish superinfection. Furthermore, we demonstrated that a single distinct allele allows strain superinfection. These results indicate that there is strong selective pressure to increase the diversity of the variant repertoire beyond what is needed for persistence within an individual host and provide an explanation, competition at the host population level, for the large genomic commitment to variant gene families in persistent pathogens.

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle of Anaplasma and Ehrlichia spp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain of Anaplasma marginale in Dermacentor andersoni as a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast, A. marginale subsp. centrale colonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless, A. marginale subsp. centrale was not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction.

Anaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginale expresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

Lifelong persistent infection of cattle is a hallmark of the tick transmitted pathogen Anaplasma marginale. Antigenic variation of Major Surface Protein 2 (MSP2) plays an important role in evasion of the host immune response to allow persistence. Antigenic variation of MSP2 is achieved by gene conversion of pseudogenes into the single operon linked expression site and the diversity of variants is defined by the pseudogene repertoire. Once an animal is persistently infected with one strain of A. marginale, infection with a second strain (superinfection) is rare. However, we recently detected animals superinfected with different strains of A. marginale and hypothesized that the msp2 pseudogene repertoire would be distinct in these superinfecting strains, consistent with encoding different sets of surface variants. Five strains of A. marginale were selected in order to identify and compare msp2 pseudogene content; these included strains with similar and different msp1alpha genotypes, and genotypes that were representative of the strains that were found in the superinfected animals. Southern blot analysis of strains associated with superinfection revealed distinctly different msp2 banding patterns, in contrast to a pattern suggesting identical pseudogene content among related strains not associated with superinfection. Indeed, targeted sequence analysis of msp2 pseudogenes showed identical pseudogene repertoires in genotypically closely related strains and varying amounts of dissimilarity in the pseudogene repertoire in strains with distinctly different msp1alpha genotypes, but totally different msp2 pseudogene repertoires between the strains that were found in superinfected animals. This finding supports the hypothesis that the occurrence of superinfection reflects the differences in the msp2 repertoire and corresponding diversity of variants.

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.

The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.

Multiple genotypically unique strains of the tick-borne pathogen Anaplasma marginale occur and are transmitted within regions where the organism is endemic. In this study, we tested the hypothesis that specific A. marginale strains are preferentially transmitted. The study herd of cattle (n = 261) had an infection prevalence of 29% as determined by competitive inhibition enzyme-linked immunosorbent assay and PCR, with complete concordance between results of the two assays. Genotyping revealed the presence of 11 unique strains within the herd. Although the majority of the individuals (70 of 75) were infected with only a single A. marginale strain, five animals each carried two strains with markedly distinct genotypes, indicating that superinfection does occur with distinct A. marginale strains, as has been reported with A. marginale and A. marginale subsp. centrale strains. Identification of strains in animals born into and infected within the herd during the period from 1998 to 2003 revealed no significant difference from the overall strain prevalence in the herd, results that do not support the occurrence of preferential strain transmission within a population of persistently infected animals and are most consistent with pathogen strain transmission being stochastic.

Whether arthropod vectors retain competence for transmission of infectious agents in the long-term absence of vector-pathogen interaction is unknown. We addressed this question by quantifying the vector competence of two tick vectors, with mutually exclusive tropical- versus temperate-region distributions, for genetically distinct tropical- and temperate-region strains of the cattle pathogen Anaplasma marginale. The tropical cattle tick Boophilus microplus, which has been eradicated from the continental United States for over 60 years, was able to acquire and transmit the temperate St. Maries (Idaho) strain of A. marginale. Similarly, the temperate-region tick Dermacentor andersoni efficiently acquired and transmitted the Puerto Rico strain of A. marginale. There were no significant quantitative differences in infection rate or number of organisms per tick following feeding on cattle with persistent infections of either A. marginale strain. In contrast, the significantly enhanced replication of the Puerto Rico strain in the salivary gland of B. microplus at the time of transmission feeding is consistent with adaptation of a pathogen strain to its available vector. However, the transmission of both strains by B. microplus demonstrates that adaptation or continual interaction between the pathogen and vector is not required for retention of vector competence. Importantly, the results clearly show that reestablishment of acaricide-resistant B. microplus in the United States would be associated with A. marginale transmission.

The most practical and reliable approach to confirming a diagnosis of OPPV or CAEV infection is a combination of serology and clinical evaluation. Although serology represents the most cost effective method of diagnosing persistently infected, clinically normal animals, testing errors occur; the frequency of error depends on the performance data of the particular serologic assay being used. When PCR detection of OPPV and CAEV becomes routinely available, this detection method can be used in rigorous eradication programs to determine the infection status of animals that cannot be definitively diagnosed by serology. The important aspects of OPPV and CAEV infection that must be considered in designing programs to prevent transmission are (1) OPPV and CAEV persist for life in the infected host, (2) a major route of transmission is to lambs and kids via colostrum and milk during nursing, (3) contact transmission among adults can occur, and (4) time variability can exist among individual sheep and goats from infection to the appearance of detectable antibodies.

Infection of horses with the hemoprotozoan Babesia equi has been reported in southern Florida, US Virgin Islands, part of Asia, Russia, India, the Middle East, Europe, Africa, Australia, South America, Central America, Mexico, Philippine Islands and some Caribbean islands. The restrictions placed on the international movement of infected horses has refocused attention on potential methods to control or eliminate infection. Don Knowles here discusses the primary chemotherapeutic compounds that have been used; the current knowledge concerning immune responses that potentially contribute to control of the parasite, and the development of infection of severe combined immuno-deficient foals as a model to dissect potential mechanisms of immunological control.

Immunization with Anaplasma marginale membranes containing major surface proteins (MSPs) induces protective immunity against clinical disease (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). For use in design of a recombinant antigen subunit vaccine for A. marginale, intermolecular relationships of known A. marginale MSPs were analyzed. Under nonreducing conditions, MSP-2 and MSP-5 occur as multimers. A large (> 300-kDa-molecular-mass), nonreduced protein complex contained MSP-1a linked by disulfide bonds to MSP-1b and by noncovalent bonds to MSP-5. MSP-2 was also noncovalently bound to this complex. The nearest neighbor membrane proteins were identified by cross-linking reactions followed by immunoblotting with anti-MSP antibodies. A cross-linked aggregate retained in the stacking gel contained MSP-1a, MSP-1b, MSP-2, MSP-3, MSP-4, and MSP-5. Collectively, the data indicate that MSP-2 and MSP-5 occur as monomers and disulfide-bonded multimers. The MSP-1 complex occurs as both disulfide-bonded and noncovalently associated MSP-1 and MSP-1b, and MSP-2 and MSP-5 are noncovalently associated with MSP-1. Also, MSP-1, MSP-2, MSP-3, and MSP-4 are nearest neighbors, and MSP-5 is noncovalently associated with this cross-linked complex.

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.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.

Advances in diagnostic assays for parasitic diseases include the use of monoclonal antibodies (MAbs) in antigen capture and competitive inhibition enzyme-linked immunosorbent assays (C-ELISA). Antigen capture ELISAs for Anaplasma marginale and Cryptosporidium parvum provide direct detection of these parasites during clinical disease, and the C-ELISA format has been adapted for detection of anti-Babesia equi, anti-A. marginale and anti-bluetongue virus antibodies. False-positive results may occur when antigen preparations in other ELISA formats are contaminated with Escherichia coli, erythrocyte or cell-culture antigens. The C-ELISA format overcomes problems of antigen purity, since the specificity of the C-ELISA depends solely on the MAb used. For this reason, the C-ELISA format is highly suited for use with recombinant antigens. Also, the use of recombinant protein in diagnostic assays precludes the need to infect animals for antigen production when the antigen cannot be produced in cell culture.

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.

Immunization with Anaplasma marginale outer membranes induced immunity against clinical disease which correlated with antibody titer to outer membrane proteins, including a 19-kDa protein (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). This 19-kDa protein, designated major surface protein 5 (MSP-5), was encoded by a single-copy 633-bp gene. The molecular mass of MSP-5, defined in immunoblots by binding to monoclonal antibody ANAF16C1, was conserved among all recognized species of Anaplasma: A. marginale, A. centrale, and A. ovis. Recombinant MSP-5, which absorbed the antibody reactivity of bovine immune serum to native MSP-5, was recognized by anti-A. marginale and anti-A. centrale immune sera in a competitive inhibition assay with monoclonal antibody ANAF16C1. The presence of antibody to the epitope defined by monoclonal antibody ANAF16C1 in all postinfection sera tested indicates that this epitope is a potential diagnostic antigen for use in identifying persistently infected cattle.

To dissect mechanisms of caprine arthritis-encephalitis lentivirus-induced arthritis, an undefined immunodominant viral glycoprotein, gp90 (G. C. Johnson, A. F. Barbet, P. Klevjer-Anderson, and T. C. McGuire, Infect. Immun. 41:657-665, 1983), was characterized. Monoclonal antibody to gp90 and specific antiserum to env gene products demonstrated that gp90 was a transmembrane protein (TM) dimer. Goats with progressive arthritis had high antibody titers to oligomeric and monomeric (38-kDa) TM.

The envelope (env) genes of two antigenic variants of caprine arthritis-encephalitis virus (CAEV), defined by serum neutralization, were expressed in vaccinia virus. Recombinant gp135 envelope glycoprotein competitively inhibited neutralizing activity of serum from CAEV-infected goats, indicating gp135 is a dominant target antigen of CAEV neutralizing antibody. In addition, type-specific neutralizing activity of goat serum directed against one variant was inhibited by both homologous and heterologous variant recombinant gp135. Hence, a CAEV variant env gene encodes type-specific neutralization epitopes of both variants. The results indicate that antigenic variation of CAEV involves env gene mutations encoding amino acid differences outside conserved neutralization epitopes affecting epitope exposure to the immune system.

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Fifteen newborn Saanen goats were orally infected with a biologically cloned isolate of caprine arthritis-encephalitis virus (CAEV-63). At 33.5 months after infection, 8 of 12 seropositive goats had CAEV-63-specific neutralizing antibody. Five neutralization-positive goats developed clinically apparent arthritis of carpal joints, three of which had periarticular swelling indicating severe synovitis. Arthritis was not evident in age-matched controls or infected goats without neutralizing antibody. Multiple viral isolates were obtained from synovial fluid or synovial fluid cells of arthritic joints between 36.4 and 44.9 months after infection, and successive isolates from individual joints were defined as antigenic variants expressing type-specific neutralization epitopes. Thus, the evolution of neutralizing antibody does not arrest viral replication or development of progressive inflammatory lesions. Rather, some antigenic variants produced in the presence of neutralizing antibody are clonally expanded within carpal joints, and severe joint inflammation is specifically associated with these variants.

A competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) was developed to detect antibody to Babesia equi. One hundred fifty-four equine serum samples from 19 countries were tested for antibody to B. equi by the complement fixation test and by CI ELISA. The CI ELISA and complement fixation test results agreed in 94% (144) of the serum samples tested. The 10 discrepant serum samples were retested and analyzed for ability to immunoprecipitate in vitro translation products from B. equi merozoite mRNA. Five discrepant results were clearly resolved in favor of the CI ELISA, and the remaining five discrepancies were not definitively resolved.

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.

The potential contributions of techniques, such as restriction enzyme analysis, nucleic acid detection, the polymerase chain reaction and competitive inhibitive tests, are only beginning to be defined. The extraordinary promise of these procedures has yet to be fully realized. However, before these techniques are accepted and widely used, they should be shown to have sensitivity and specificity comparable to those of current tests. Finally, they should be safe, easy to conduct and automated to facilitate the study of large numbers of specimens.