Animal pathogens and their impact on animal health, the economy, food security, food safety and public health.

Animal pathogens attract attention in both the livestock and public health sectors for their impacts on socio-economics, food safety and security, and human health. These impacts are felt at the household, national, regional and global levels. Whereas the World Organisation for Animal Health (OIE) has identified 118 animal diseases as notifiable, based on their potential for impact on trade, there is a selected subset that have been classified as posing a greater threat to countries due to unique characteristics, such as being highly transmissible, spreading rapidly within and between countries, and requiring cooperation between several countries to control their spread or exclude them. While these 'transboundary diseases' are endemic in much of the world, particularly the developing nations, many countries are classified as disease free. Following the terrorist events of 11 September 2001 in the United States, a small group of zoonotic pathogens and a group of animal-specific pathogens (those that cause what are referred to as `high-consequence foreign animal diseases'), were classified as high-risk, biothreat 'select agents'. Rather than providing a comprehensive review of all animal pathogens, the authors briefly review the impact of these high-risk biothreat agents on animal health, the economy, food security and safety, and public health, using highly pathogenic avian influenza, foot and mouth disease and brucellosis as examples. They focus on the impact of these diseases in the context of high-income countries and low- and middle-income countries, comparing and contrasting their impact at the national and individual household levels.

Les agents pathogènes d'origine animale revêtent une grande importance tant pour le secteur de l'élevage que pour celui de la santé publique, en raison de leurs conséquences sur la société et l'économie, sur la sécurité alimentaire, sur la sécurité sanitaire des aliments et sur la santé publique. Ces impacts sont perceptibles à l'échelle des ménages, des pays, des régions et du monde. L'Organisation mondiale de la santé animale (OIE) a établi une liste de 118 maladies animales à déclaration obligatoire en se basant principalement sur leurs conséquences potentielles pour le commerce ; néanmoins, un sous-ensemble de la liste concerne les maladies qui font peser un risque élevé sur les pays, de par leurs caractéristiques uniques, par exemple leur contagiosité, la rapidité de leur potentiel de propagation dans le territoire national ou d'un pays à l'autre, ou la nécessité de mettre en place une coopération internationale en vue de maîtriser leur propagation ou de les éliminer. Ces « maladies transfrontalières ¼ sont présentes à l'état endémique dans une grande partie du monde, en particulier dans les pays en développement, tandis que d'autres pays sont considérés comme « indemnes ¼. Suite aux attaques terroristes du 11 septembre 2001 aux États-Unis d'Amérique, les maladies animales dites « exotiques ¼ ainsi qu'un petit nombre d'agents pathogènes zoonotiques ont été classés dans la catégorie des « agents biologiques à haut risque ¼. Plutôt que de fournir un inventaire exhaustif des agents pathogènes d'origine animale, les auteurs résument l'impact de ces agents biologiques à haut risque sur la santé animale, l'économie, la sécurité alimentaire, la sécurité sanitaire des aliments et la santé publique, en illustrant leur propos avec les exemples de l'influenza aviaire hautement pathogène, la fièvre aphteuse et la brucellose. Ils examinent l'impact de ces maladies dans le contexte des pays à revenus élevés, mais aussi des pays à revenus faibles ou intermédiaires, en comparant et en détaillant les impacts respectifs à l'échelle nationale ainsi qu'à l'échelle des ménages.

Por su influencia en factores socioeconómicos y en temas como la higiene de los alimentos, la seguridad alimentaria o la salud humana, los patógenos animales atraen la atención de los sectores de la producción animal y la salud pública. Dicha influencia se deja sentir tanto a nivel de los hogares como a escala nacional, regional y mundial. Aunque la Organización Mundial de Sanidad Animal (OIE) tiene catalogadas 118 enfermedades animales como «de declaración obligatoria¼, atendiendo a sus posibles consecuencias para el comercio, hay un pequeño subconjunto de ellas que se consideran especialmente peligrosas para los países porque revisten características singulares, como el hecho de ser muy transmisibles, propagarse con gran rapidez entre los países y dentro de ellos o exigir cooperación entre varias naciones para combatir su propagación o atajarlas. Estas «enfermedades transfronterizas¼ son endémicas en gran parte del mundo, especialmente en las naciones en desarrollo, pero también hay muchos países que están considerados «libres¼ de ellas. Después de los atentados terroristas que sufrieron los Estados Unidos el 11 de septiembre de 2001, las llamadas enfermedades animales «extranjeras¼, junto con un pequeño grupo de patógenos zoonóticos, fueron catalogadas como «agentes selectos¼ de alto riesgo de amenaza biológica. En lugar de ofrecer una panorámica completa de todos los patógenos animales, los autores repasan brevemente el impacto de estos agentes calificados de alto riesgo y portadores de una amenaza biológica en la sanidad animal, la economía, la seguridad alimentaria, la higiene de los alimentos y la salud pública, valiéndose para ello de los ejemplos de la influenza aviar altamente patógena, la fiebre aftosa y la brucelosis. Centrándose en el impacto de estas enfermedades en el contexto de los países de renta alta y en el de los países de renta baja o media, comparan y contrastan tal impacto a escala nacional y en el ámbito de los hogares.

A survey of veterinary antimicrobial prescribing practices, Washington State 2015.

Antimicrobial resistance is a growing global health issue. It is also a recognised problem in veterinary medicine. Between September and December 2015 the authors administered a cross-sectional survey to licensed veterinarians in Washington State to assess factors affecting antimicrobial prescribing practices among veterinarians in Washington State. Two hundred and three veterinarians completed the survey. The majority of respondents (166, 82 per cent) were engaged in small animal or exotic animal practice. 24 per cent of respondents reported not ordering culture and sensitivity (C/S) testing in practice. Of the 76 per cent of veterinarians who reported ordering C/S tests, 36 per cent reported ordering such testing 'often' or 'always' when treating presumptive bacterial infections. Most respondents (65 per cent) mentioned cost as the most common barrier to ordering a C/S test. Only 16 (10 per cent) respondents reported having access to or utilising a clinic-specific antibiogram. This survey demonstrated that while antimicrobials are commonly used in veterinary practice, and veterinarians are concerned about antimicrobial resistance, cost is a barrier to obtaining C/S tests to guide antimicrobial therapy. Summaries of antimicrobial resistance patterns are rarely available to the practising veterinarian. Efforts to promote antimicrobial stewardship in a 'One Health' manner should address barriers to the judicious use of antimicrobials in the veterinary practice setting.

Enhanced B-lymphocyte expression of IL-2R alpha associated with T lymphocytosis in BLV-infected persistently lymphocytotic cows.

Peripheral blood lymphocytes from bovine leukemia virus (BLV)-negative and BLV-infected, aleukemic cows with persistent lymphocytosis were evaluated for expression of B and T lymphocyte subset-specific molecules and co-expression of the interleukin-2 receptor alpha (IL-2R alpha) molecule. Results demonstrate enhanced mitogen-induced expression of the IL-2R alpha molecule on B lymphocytes from BLV-infected, lymphocytotic cows. Lymphocyte subset analyses further demonstrate that BLV-infected, lymphocytotic cows are not only characterized by sustained elevations in CD5+ B lymphocytes, but also show significantly elevated numbers of CD3+, CD4+, and CD8+ T lymphocytes. These results provide evidence suggesting that B lymphocytes from BLV-infected, lymphocytotic cows are more sensitive to activation signals and up-regulation of the IL-2 signaling pathway than lymphocytes from clinically normal BLV-free cows, and that T lymphocytes may be involved in the aberrant regulatory pathways underlying BLV-induced persistent B lymphocytosis.

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.

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.

Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins.

Monoclonal antibodies binding to the surface of live Mexico isolate Babesia bigemina merozoites have defined 4 parasite-encoded surface antigens (36, 45, 55, and 58 kDa) that are potential targets for immune-mediated neutralization of merozoites. In this study, we have characterized the post-translational modification, antigenic polymorphism, and immunogenicity of these 4 proteins. Monoclonal antibody immunoaffinity-purified 36- and 55-kDa polypeptides were identical in gel electrophoresis to immunoprecipitated radiolabeled proteins, while the purified 45-kDa protein consisted of 2 closely spaced polypeptides with relative molecular weights of 45 and 43 kDa. The 36-, 45-, and 55-kDa proteins were post-translationally modified by incorporation of [3H]glucosamine and [3H]myristic acid, suggesting they are integral membrane proteins secured by a phosphatidylinositol anchor. Cross-reactivity studies with monoclonal and monospecific polyclonal antibodies revealed marked antigenic polymorphism of these 3 glycoproteins among diverse geographic isolates. In contrast, none of the polypeptides bound by anti-p58 monoclonal antibody were glycosylated or myristilated. Both monoclonal and monospecific polyclonal antibodies recognizing p58 bound to similar molecular weight proteins in 4 additional isolates of B. bigemina from Mexico, Puerto Rico, St. Croix, and Kenya, suggesting widespread conservation of p58 immunogenic epitopes among geographic isolates. Calves immunized with immunoaffinity purified gp45, gp55, or p58 antigens were able to neutralize the infectivity of merozoites as indicated by significant reductions in the peak parasitemia after experimental challenge. Precise definition and appropriate presentation of neutralization sensitive epitopes on gp45, gp55, or p58 may enhance the merozoite neutralizing immune response in immunized 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.

Isolation, sequence and differential expression of the p58 gene family of Babesia bigemina.

Four copies of the gene encoding the merozoite surface protein p58 from the protozoan hemoparasite Babesia bigemina were amplified from genomic DNA by polymerase chain reaction (PCR) techniques, molecularly cloned and subjected to DNA sequence analysis. The amplified DNA (Bbg7, Bbg9, Bbg13, Bbg14) could be placed into 2 classes with respect to its size and the length of the open reading frame (ORF). With the exception of a single base substitution, the sequence of Bbg13 is identical to the cDNA sequence published earlier [1]. The Bbg7 and Bbg14 copies of p58 diverged from Bbg13 sequence at regions towards the 3' and 5' ends, respectively. In contrast, Bbg9 has incorporated both regions of divergence within its sequence. Using a cloned strain of B. bigemina, RNA-PCR and Northern blot analyses demonstrate the in vivo transcription of 3 of the 4 copies, although one of the 3 expressed copies is present in very low abundance. The relative abundance and size of the two p58 mRNA species detected are consistent with the 58- and 55-kDa proteins detected by in vitro translation of B. bigemina poly(A)+ mRNA by immunoprecipitation with an anti-p58 monospecific antibodies. These results indicate that the gene encoding p58 exists as a multigene family that appears to be differentially expressed in the blood stage of the parasite's life cycle.

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.

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.

Immunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface protein.

The gene encoding the conserved, neutralization-sensitive surface protein p58 of Babesia bigemina was cloned and sequenced. An open reading frame of 1440 bases was found to encode a protein with a predicted size of 54 kDa. A transmembrane hydrophobic domain and signal peptide were present at the amino-terminus. The polypeptide encoded by a nearly full length cDNA was expressed in bacteria and contained epitope(s) reactive with anti-p58 polyclonal and monoclonal antibodies. Serum antibodies from rabbits immunized with a lysate of recombinant bacteria specifically immunoprecipitated native p58 from [35S]methionine-labeled B. bigemina antigens. In addition, the sera contained antibodies that bound to the surface of live merozoites from 4 geographically different Latin American isolates, confirming the presence and immunogenicity of conserved, surface-exposed epitopes on the recombinant polypeptide. This molecular clone will now enable immunization trials in cattle designed to better evaluate the ability of p58 to induce immune protection by vaccinating with constructs containing only conserved, neutralization-sensitive epitopes.

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.

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.

Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice.

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon-gamma; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon-gamma. Mice treated with anti-interferon-gamma alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon-gamma-treated mice was associated with antigen-specific antibody IgG1 > IgG2a and a three-fold decreased ratio of antigen-specific interferon-gamma:interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a > IgG1 and a three-fold increased ratio of antigen-specific interferon-gamma:interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon-gamma, as indicated by concomitant in vivo interferon-gamma neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon-gamma, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.

Screening of a Babesia bigemina cDNA library with monoclonal antibodies directed to surface antigens.

A Babesia bigemina cDNA library prepared in lambda ZAP bacteriophage vector was immunoscreened to detect clones expressing surface-exposed epitopes of B. bigemina. A nonradioactive indirect plaque-lift immunoassay was used to detect the positive clones. The primary antibody consisted of a pooled sample of six monoclonal antibodies (mAb) specific for B. bigemina that recognizes various parasite surface antigens of different molecular mass. Screening of approximately 300,000 plaque-forming units from the lambda ZAP cDNA expression library resulted in the identification of five positive clones. The five recombinant clones were immunoscreened individually with each of the six mAb. All five independently obtained clones consisted of lambda ZAP recombinants expressing B. bigemina components recognized by mAb C2F3G3 and B1B3C4. Restriction enzyme digests of rescued recombinant phagemids showed that only four clones contained B. bigemina cDNA. One clone (lambda ZAP Bbi1) contained an insert of approximately 0.6 kBp whereas the other three clones (lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5) carried a cDNA insert of approximately 1.7 kBp. Immunoblotting of protein extracts from recombinants lambda ZAP Bbi2, lambda ZAP Bbi3, and lambda ZAP Bbi5 with mAb C2F3G3 and B1B3C4 demonstrated the expression of a recombinant B. bigemina polypeptide of 55 kDa in E. coli.

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

Heterologous strain challenge of cattle immunized with Anaplasma marginale outer membranes.

In this study, we tested the hypothesis that outer membrane immunization would induce protection against an antigenically variant strain. Outer membranes were isolated from the Virginia strain of Anaplasma marginale using density gradient centrifugation, combined with saponin adjuvant, and used to immunize Friesian cattle in Zimbabwe. Immunized cattle developed high antibody titers (80,000-160,000) against outer membrane polypeptides including MSP-2 and MSP-5 in both the homologous Virginia and heterologous Zimbabwe strains. Outer membrane immunized cattle were protected significantly following challenge with 10(4) Zimbabwe strain parasitized erythrocytes, demonstrated by significant differences in prepatent period and peak rickettsemia compared with adjuvant immunized control cattle. One outer membrane immunized animal was completely protected against infection. However, there were no overall significant differences in severity of anemia between cattle immunized with outer membrane and the control group, indicating that a significant reduction in rickettsemia does not necessarily result in less severe anemia.