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.

Immunization with an isolate-common surface protein protects cattle against anaplasmosis.

Hemoparasitic diseases are endemic in half the world's livestock production areas and are the greatest obstacle to improved meat, milk, and fiber production in the Third World. The most prevalent of these diseases, anaplasmosis, occurs throughout tropical and subtropical regions and is responsible for 50,000 to 100,000 cattle deaths annually in the United States alone. Despite its prevalence and the severity of the losses, an effective immunoprophylaxis for anaplasmosis has not been developed. A neutralization-sensitive epitope on a surface protein with a molecular weight of 105,000 (Am 105) of the causative rickettsia Anaplasma marginale was identified by monoclonal antibody inhibition of infectivity. This epitope was determined to be common to eight isolates with antigenic, morphologic, and protein structural differences. Cattle immunized with Am 105 purified by immunoaffinity chromatography were protected against challenge with virulent Anaplasma marginale. The identification of Am 105 as bearing isolate-common epitopes capable of inducing protection in immunized cattle provides the basis for the development of an effective subunit vaccine for bovine anaplasmosis.

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.

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 (

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.

Association of GP51 expression and persistent CD5+ B-lymphocyte expansion with lymphomagenesis in bovine leukemia virus infected sheep.

Alterations in the circulating CD5+ B-lymphocyte population, in vitro GP51 expression, and in vivo tax/rex expression that may precede lymphomagenesis were characterized prospectively in ten experimentally BLV-infected sheep. Infection with pathogenetic BLV resulted in a significant expansion of the circulating CD5+ B-lymphocyte population in six infected sheep. Of the remaining four infected sheep that did not have persistently elevated CD5+ B-lymphocyte counts, three developed lymphoid neoplasia within 14 months post-inoculation. Neoplastic cells from two of these three sheep were CD5- B-lymphocytes, while cells from the third were CD5+ B-lymphocytes. In vitro GP51 expression was a consistent feature of circulating lymphocytes from all three sheep developing tumors, but high level tax/rex gene transcription was not detected in circulating lymphocytes prior to lymphomagenesis. Neither in vitro GP51 expression nor high level tax/rex gene transcription was associated with expansion of the CD5+ B-lymphocyte population in sheep with significantly elevated CD5+ B-lymphocyte counts. These observations indicate that BLV infection in sheep results in expansion of the circulating CD5+ B-lymphocyte population, and that this expansion is not required for the subsequent development of BLV-associated lymphoid neoplasia.

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

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.

Immunization with Babesia bigemina rhoptry-associated protein 1 induces a type 1 cytokine response.

Rhoptry-associated protein-1 (RAP-1) homologues of Babesia bigemina and Babesia bovis are promising candidates for inclusion in subunit vaccines against these hemoprotozoan parasites. Partial protection against challenge infection has been achieved with native forms of these antigens, but the mechanism of immunity has not been thoroughly defined. We previously demonstrated that a panel of antigen-specific T helper cell clones derived from B. bigemina RAP-1-immunized cattle expressed relatively high levels of interferon-gamma (IFN-gamma) protein and transcript and low levels of interleukin-4 (IL-4), indicative of a type 1 immune response. In the current study we present evidence that subcutaneous immunization with native B. bigemina RAP-1 protein in RIBI adjuvant induces a predominant type 1 immune response in vivo, characterized by relatively high levels of IFN-gamma and IL-2 and low levels of IL-4 and IL-10 mRNA in the draining prescapular lymph node. Ex vivo restimulation of draining lymph node lymphocytes with specific antigen resulted in proliferation and enhanced expression of IL-2 and IFN-gamma, whereas IL-4 and IL-10 transcript levels remained relatively low. These findings show that our previously described cytokine profiles of antigen-specific cloned T cell lines are representative of autologous in vivo responses and confirm that type 1 recall responses to B. bigemina RAP-1 can be evoked in immunized animals by native parasite antigen.

Genetic variation in the dimorphic regions of RAP-1 genes and rap-1 loci of Babesia bigemina.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle. RAP-1 has two regions of sequence dimorphism at the carboxy and amino terminal ends, respectively. Neutralization-sensitive, surface-exposed B-cell epitopes are present in the amino terminal variant type 1 (NT-1), and CD4+ T-cell epitopes in the carboxy terminal variant type 1 (CT-1). Importantly, antibodies recognizing NT-1 epitopes do not cross react with NT-2 and CD4+ T-cells recognizing epitopes in CT-1 do not cross react with CT-2, suggesting that variation in dimorphic regions of RAP-1 is immunologically significant. We evaluated rap-1 locus structure and the extent of sequence variation in the dimorphic regions of rap-1 genes from geographically diverse strains of B. bigemina. All strains contained NT-1 and NT-2 the encoding sequences were highly conserved, with at least 99%, nucleotide identity among strains. However, the Puerto Rico strain encoded a hybrid NT-1/NT-2 sequence which appears to have originated by a gene conversion event. The 3' ends of rap-1 genes, which include the carboxy terminal variants, are conserved among strains. A new and conserved CT variant (CT-3), with a region of sequence identity to CT-2 and a sequence not related to either CT-1 or CT-2, was identified in all strains of B. bigemina. All but one strain encode both NTs and the three CT variants. The S1A strain, an attenuated strain from Argentina, does not encode CT-2. While NT-1 is associated only with CT-1, NT-2 can be associated with all three CT variants in RAP-1. Within the genome, rap-1 genes are arranged in tandem repeats but with different gene copy number and arrangements among strains. Collectively, the data suggest that gene conversion and unequal recombination events contribute to overall rap-1 sequence conservation among gene variants and strains but may also generate new rap-1 variants.

Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus.

The complexity of multigene families encoding rhoptry proteins and the generation of new variants in these families are constraints to development of vaccines incorporating rhoptry proteins. For example, the Babesia bigemina rhoptry associated protein (rap)-1 locus is composed of tandemly arranged genes including four polymorphic rap-1a genes and two classes of divergent genes, rap-1b and rap-1c. B. bigemina rap-1 polymorphism reflects recombination and gene conversion and results in multiple RAP-1 proteins with unique B- and T-cell epitopes. Is this complex locus structure and recombination a required feature of the rap-1 gene family among Babesia species? We addressed this question by analysis of the rap-1 locus in B. bovis. Sequence analysis of an 11 kb genomic clone representing the B. burn rap-1 locus revealed only two identical and continuous rap-1a gene copies, rap 1a-1 and rap-1a-2, located in a similar head to tail orientation. Using the conserved ig gene as a marker for the 3' boundary of the rap-1 locus, we conclude that divergent rap-1b and rap-1c genes, present in B. bigemina, are not similarly cis-linked to the B. bovis rap-1 locus. Analysis of the rap-1a genes 1 and 2 from each of multiple B. bovis strains from North and South America demonstrated RAP-1 size conservation with very limited amino acid sequence variation. The results suggest that the simple two gene arrangement in the B. bovis rap-1 gene family was generated by gene duplication and, in contrast to the B. bigemina rap-1 locus, both genes evolved together using homogenization mechanisms with point mutation as the single mechanism for gene variation. Three discontinuous non-rap-1 genes are closely cis-linked to the B. bovis rap-1 locus and the presence of multiple introns in these genes may limit rap-1 gene variation due to unequal crossing over. The different mechanisms likely involved in the evolution of the rap-1 family in B. bigemina versus B. bovis are reflected in the marked structural and antigenic polymorphism in the B. bigemina RAP-1 molecules as compared with the essentially monomorphic RAP-1 in B. bovis.

Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family.

Monospecific antibodies against native and recombinant versions of the major merozoite surface antigen (MSA-1) of Babesia bovis neutralize the infectivity of merozoites from Texas and Mexico strains in vitro. Sequence analysis shows that MSA-1 and a related, co-expressed 44 kDa merozoite surface protein (MSA-2) are encoded by members of a multigene family previously designated BabR. BabR genes, originally described in Australia strains of B. bovis, are notable because their marked polymorphism is apparently mediated by chromosomal rearrangements, but protein products of BabR genes have not previously been identified. The 3' terminal 173 nucleotides of the MSA-1 gene, including 60 nucleotides of untranslated sequence, are highly similar to the 3' terminal sequences of BabR 0.8 (84% identity) and MSA-2 (94% identity). Alignment of the predicted protein sequences demonstrates significant overall homology between MSA-1 and MSA-2, and between both proteins and the amino terminal BabR sequence. MSA-1 nucleic acid probes also hybridize weakly to genomic DNA from the Australia 'L' strain, even though this strain does not express merozoite surface epitopes cross-reactive with MSA-1 or MSA-2. Hybridization of these same probes to genomic DNA from the cloned Mexico strain reveals a pattern of bands compatible with two copies each of MSA-1 and MSA-2. Proteins encoded by this B. bovis gene family have been designated variable merozoite surface antigens (VMSA). The extent and mechanism of VMSA polymorphism among strains will be important when evaluating the role these surface proteins have in the host-parasite interaction, including immunity to blood stages.

Dimorphic sequences of rap-1 genes encode B and CD4+ T helper lymphocyte epitopes in the Babesia bigemina rhoptry associated protein-1.

The rhoptry-associated protein-1 (RAP-1) of Babesia bigemina induces protective immune responses in cattle and contains neutralization-sensitive B cell epitopes. RAP-1 variants containing blocks of sequence dimorphism in the amino and carboxy terminal ends are encoded by four nonallelic genes in B. bigemina. Epitopes recognized by RAP-1 specific monoclonal antibodies (MAbs) and bovine CD4+ T cell clones were mapped to determine whether these epitopes are localized in the amino and carboxy terminal dimorphic regions. Four B cell epitopes, including a neutralization-sensitive epitope, required both the amino terminal variant type 1 (NT-1) and non-dimorphic sequences for conformation. Intrachain disulfide bonds were required for at least one of these epitopes, since reduction and alkylation of cysteine residues abolished MAb binding. A fifth B cell epitope was mapped to the carboxy terminal variant type 1 (CT-1). As expected, the neutralizing MAb and two other MAbs requiring NT-1 for epitope binding recognized only the two RAP-1 variants with the NT-1 sequence, while the MAb binding an epitope in CT-1 did not bind RAP-1 variants with CT-2. In contrast, the fourth MAb requiring NT-1 for binding recognized all rap-1 gene products, indicating that dimorphic residues are not part of the epitope recognized by this MAb. Bovine CD4+ T cell clones characterized previously as responding in a strain dependent fashion recognized at least one epitope in CT-1, and did not cross-react with CT-2. A second group of bovine CD4+ T cell clones that responded to multiple parasite strains recognized an epitope in a non-dimorphic region of RAP-1. These data indicate that dimorphic regions of RAP-1 encode unique B and T helper lymphocyte epitopes and may be required for enhanced protective immune responses in cattle.

Molecular characterization of Babesia bovis merozoite surface proteins bearing epitopes immunodominant in protected cattle.

Eight surface-radioiodinated merozoite proteins from a cloned, pathogenic isolate of Babesia bovis can be immunoprecipitated by antibody from cattle that are completely protected against clinical babesiosis. Among these eight surface proteins, the 55- and 42-kDa molecules are biosynthetically labeled with [3H]glucosamine. The 42-kDa glycoprotein can also be labeled with [3H]myristic acid and partitions exclusively into the detergent phase in Triton X-114 extracts, indicating that it is an integral membrane protein and suggesting that it is anchored by a glycosylphosphatidylinositol moiety. Antibody-mediated protection against B. bovis merozoites most probably requires a high level of circulating antibody to ensure antibody-merozoite binding during the parasite's brief extra-erythrocytic phase. Antibodies in diluted sera selectively recognize the 120-, 85-, 55- and 42-kDa surface proteins. Only the 42-kDa integral membrane protein is reactive with serum antibodies diluted greater than or equal to 1:16,000. Thus, we hypothesize that these immunodominant proteins, especially the transmembrane 42-kDa glycoprotein, are important to the induction of the protective immune response and are candidates for an improved vaccine against babesiosis.

Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes.

A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain of Babesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specifically immunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [35S]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia 'L', and Texas strains of B. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia 'L' B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis.

Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis.

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i-RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis.

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1.

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Strategies to interrupt the development of Anaplasma marginale in its tick vector. The effect of bovine-derived antibodies.

Anaplasma marginale is a rickettsia transmitted by ticks that invades and multiplies in bovine erythrocytes causing the disease anaplasmosis. A complex developmental cycle occurs within ticks that begins in midgut cells, with subsequent infection in gut muscle cells. Final development occurs in salivary glands from where the rickettsia is transmitted to the vertebrate host. At each site of development, A. marginale multiplies within membrane-bound inclusions. Attempts to control anaplasmosis have focused on cattle and have included immunization and prophylactic treatment with tetracyclines. New strategies for control of anaplasmosis are being focused on the tick vector. Development of vaccines against hemoparasites in ticks may be feasible because vertebrate host immunoglobulins appear to cross the midgut epithelium of invertebrates and enter the hemolymph without breakdown. We tested the effect of A. marginale antibodies ingested by ticks with the bloodmeal on infections in ticks. Cattle were immunized with purified outer membrane proteins of erythrocytic-derived parasites. Infections in ticks exposed to the immunized cattle were determined using an Anaplasma-specific DNA probe, light and electron microscopy, and tick transmission studies. Vaccine-derived antibodies did not appear to affect the development and transmission of A. marginale in ticks. Further studies are needed to determine if bovine antibodies remain intact within ticks and whether the tick stage of A. marginale has unique surface antigens from the erythrocytic stage.

Immunity to Rhodococcus equi.

Rhodococcal pneumonia is an important, life threatening disease of foals and immunosuppressed humans. Increased knowledge of the mechanisms of protective immunity are required in order to develop an effective immunoprophylaxis strategy for horses and immunotherapeutic regiments for people. Both humoral and cellular components of the immune system may be involved in immune clearance of R. equi. The susceptibility of foals less than 4-6 months of age is postulated to reflect waning maternal antibody, and passive transfer of hyperimmune plasma can provide protection on endemic farms. However, effective clearance is likely to require appropriate cellular responses, including the secretion of cytokines. In murine models, both CD4+ and CD8+ T lymphocytes can reduce bacterial counts in the lung. CD4+ cells appear to be both required and sufficient, and IFN-gamma is a primary mediator. Clearance appears to be a type 1 immune response while type 2 responses may lead to a failure to clear and lesion development. It remains to be determined how the cellular immunity experiments reported in mice relate to horses and humans. Likewise, the role of specific R. equi antigens in protective immunity has not been determined.

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.