Informal sale of antibiotics in Guatemalan convenience stores before and after implementation of federal antibiotic dispensing legislation.

BACKGROUND: Convenience stores in Guatemala provide essential consumer goods in communities, but many dispense antibiotics illegally. Federal legislation, passed in August of 2019, requires prescriptions for antibiotic purchase at pharmacies but it is unclear if this legislation is enforced or if it has any impact on unlawful sales of antibiotics. METHODS: To determine if antibiotic availability changed in convenience stores, we carried out a repeated measures study collecting antibiotic availability data before and after implementation of the dispensing regulation. RESULTS: There was no statistical difference in the proportion of convenience stores that sold antibiotics before and after antibiotic regulations [66.6% (295/443) and 66.7% (323/484), respectively, P>0.96], nor in the number of stores selling amoxicillin [55.5% (246/443) and 52.3% (253/484), respectively, P>0.96], but fewer stores (20%) sold tetracycline capsules after regulation was passed (P<0.05). For stores visited both before and after passage of legislation (n=157), 15% stopped selling antibiotics while 25% started selling antibiotics. Antibiotics from convenience stores were reportedly sold for use in people and animals. CONCLUSIONS: Antibiotics remain widely available in convenience stores consistent with no significant change in the informal sector after implementation of prescription requirements for pharmacies. Importantly, effects from regulatory change could have been masked by potential changes in antibiotic use during the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic.

Sir Arnold Theiler and the discovery of anaplasmosis: a centennial perspective.

Sir Arnold Theiler's research in 1908/09 led to the discovery of the first rickettsial pathogen, Anaplasma marginale, and set the stage for his development and implementation of an effective live vaccine based on a less virulent strain, A. marginale ss. centrale. His 1910 report, describing A. marginale, is among the classic monographs in infectious disease research, presenting not only observations in exacting detail but also highlighting the deductive reasoning leading to association of a new pathogen with a specific disease. With a centennial perspective and both conceptual frameworks and molecular tools unimaginable in Theiler's time, the significance of several observations in the original report--cyclic bacteremia, strain superinfection, and taxonomic position--is now clear and highlight the broad applicability of key principles of pathogen biology.

A commentary on domestic animals as dual-purpose models that benefit agricultural and biomedical research.

Research on domestic animals (cattle, swine, sheep, goats, poultry, horses, and aquatic species) at land grant institutions is integral to improving the global competitiveness of US animal agriculture and to resolving complex animal and human diseases. However, dwindling federal and state budgets, years of stagnant funding from USDA for the Competitive State Research, Education, and Extension Service National Research Initiative (CSREES-NRI) Competitive Grants Program, significant reductions in farm animal species and in numbers at land grant institutions, and declining enrollment for graduate studies in animal science are diminishing the resources necessary to conduct research on domestic species. Consequently, recruitment of scientists who use such models to conduct research relevant to animal agriculture and biomedicine at land grant institutions is in jeopardy. Concerned stakeholders have addressed this critical problem by conducting workshops, holding a series of meetings with USDA and National Institutes of Health (NIH) officials, and developing a white paper to propose solutions to obstacles impeding the use of domestic species as dual-purpose animal models for high-priority problems common to agriculture and biomedicine. In addition to shortfalls in research support and human resources, overwhelming use of mouse models in biomedicine, lack of advocacy from university administrators, long-standing cultural barriers between agriculture and human medicine, inadequate grantsmanship by animal scientists, and a scarcity of key reagents and resources are major roadblocks to progress. Solutions will require a large financial enhancement of USDA's Competitive Grants Program, educational programs geared toward explaining how research using agricultural animals benefits both animal agriculture and human health, and the development of a new mind-set in land grant institutions that fosters greater cooperation among basic and applied researchers. Recruitment of outstanding scientists dedicated to using domestic animal models for agricultural and biomedical research, strong incentives for scientists to take advantage of training opportunities to write NIH grants, and greater NIH and USDA cooperation to sponsor the use of agricultural animals as dual-purpose animal models that benefit agriculture and biomedicine will also be necessary. In conclusion, the broad diversity of animal models needed for agricultural and biomedical research is at risk unless research priorities at the land grant universities are critically evaluated and financial support for such research is dramatically increased.

Influence of corn size distribution on the diastatic power of malted barley and its impact on other malt quality parameters.

Detailed studies were carried out on the influence of corn size distribution on the values obtained for diastatic power (DP) of commercially malted barley. Malted barley was screened using a screening box, and the DP activities of the different corns retained on the different compartments of the screening box were determined. The malt samples retained on the 2.8 mm screen had the highest DP activity, whereas the small corns (

In vivo endothelial cell infection by Anaplasma marginale.

Anaplasma marginale has recently been shown to infect endothelial cells in vitro, but it remains unknown as to whether endothelial infection also occurs in vivo. In this report, we demonstrate through dual fluorescence microscopy that A marginale, detected by the monoclonal antibody ANAF16C1, co-localizes with the endothelial cell marker, von Willebrand factor, in tissue sections from an experimentally inoculated calf. The results indicate that A marginale infection includes endothelial cells and has implications for both pathogenesis and immune mechanisms.

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Detection of the Anaplasma centralevaccine strain and specific differentiation from Anaplasma marginale in vaccinated and infected cattle.

Bovine anaplasmosis caused by the intraerythrocytic rickettsia Anaplasma marginale is the most prevalent tick-borne disease of cattle worldwide. The most efficient method to control anaplasmosis is by vaccination using live Anaplasma centrale, a closely related species or subspecies of low pathogenicity that is capable of inducing significant protection against the more virulent A. marginale. In the present study, we applied PCR assays to detect and discriminate field infection with A. marginale from A. centrale persistently infected vaccinates. Direct and one-stage nested PCR were based on A. centrale mbp58 specific sequence, with the assay sensitivity level of 0.00001% for nested PCR performed in a single amplification step. Size polymorphism in the A. marginale msp1 alpha gene among strains was used to design a PCR capable of discriminating between the Israel T and NT strains of A. marginale and the encoded MSP1a size polymorphism was confirmed by immunoprecipitation. The detection of A. centrale in 72% of vaccinated field-grazing cattle clearly indicated that the majority of vaccinated cattle remain carriers. A. marginalewas detected in 64% of these vaccinated cattle, demonstrating that, as expected, natural transmission occurs within the endemic region. The lack of severe A. marginaleoutbreaks in this region, despite ongoing transmission, is consistent with protection being provided by widespread vaccination with A. centrale.

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.

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 unique phospholipid organization in bovine erythrocyte membranes.

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

Molecular basis for variable expression of merozoite surface antigen gp45 among American isolates of Babesia bigemina.

Immunization with the merozoite surface glycoprotein gp45 induces protection against challenge using the homologous Babesia bigemina strain. However, gp45 B-cell epitopes are highly polymorphic among B. bigemina strains isolated from different geographical locations within North and South America. The molecular basis for this polymorphism was investigated using the JG-29 biological clone of a Mexico strain of B. bigemina and comparison with the Puerto Rico, St. Croix, and Texcoco strains. The molecular size and antibody reactivity of gp45 expressed by the JG-29 clone were identical to those of the parental Mexico strain. gp45 cDNA and the genomic locus encompassing gp45 were cloned and sequenced from JG-29. The locus sequence and Southern blot data were consistent with a single gp45 copy in the JG-29 genome. The JG-29 cDNA expressed the full-length protein recognized by the gp45-specific monoclonal antibody 14/1.3.2. The genomes of the Puerto Rico and St. Croix strains of B. bigemina were shown to lack a closely related gp45-like gene by PCR using multiple primer sets and by Southern blots using both full-length and region-specific gp45 probes. This genomic difference was confirmed using unpassaged isolates from a 1999 disease outbreak in Puerto Rico. In contrast, the Texcoco strain retains a gp45 gene, encoding an open reading frame identical to that of JG-29. However, the Texcoco gp45 gene is not transcribed. These two mechanisms, lack of a closely related gp45-like gene and failure to transcribe gp45, result in generation of antigenic polymorphism among B. bigemina strains, and the latter mechanism is unique compared to prior mechanisms of antigenic polymorphism identified in babesial parasites.

Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses.

Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.

Characterization of caprine interleukin-4.

Caprine interleukin-4 (IL-4) cDNA was cloned from RNA of mitogen stimulated goat peripheral blood mononuclear cells utilizing reverse transcriptase-polymerase chain reaction. The sequence of caprine IL-4 cDNA corresponds to a 535 nucleotide mRNA with 5'- and 3'-untranslated regions and a 405 nucleotide open reading frame, the first 66 nucleotides of which encode a putative signal peptide. Mature IL-4 is a 12.8kDa protein containing six cysteine residues and two potential N-linked glycosylation sites and is highly homologous with other ruminant IL-4. The predicted molecular mass of mature unglycosylated IL-4 was confirmed by western blot of recombinant caprine IL-4 expressed in bacteria with a monoclonal antibody against a carboxyterminal peptide derived from the predicted amino acid sequence of bovine IL-4. Eukaryotic expression plasmids containing caprine IL-4 cDNA were used to characterize recombinant IL-4. Transcription of IL-4 mRNA was confirmed by transfection of COS-7 and goat synovial membrane cells, and recombinant IL-4 produced by stably transfected L929 cells inhibited inducible nitric oxide synthase in macrophages. Genetic immunization of mice with a caprine IL-4 cDNA expression plasmid induced antibodies against recombinant caprine IL-4 produced in bacteria.

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.

Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission.

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.

Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.

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.

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Identification of fetal liver tyrosine kinase 3 (flt3) ligand domain required for receptor binding and function using naturally occurring ligand isoforms.

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.