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.

Prospects for recombinant vaccines against Babesia bovis and related parasites.

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

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.

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.

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

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.

Immunization of BALB/c mice with killed Neospora caninum tachyzoite antigen induces a type 2 immune response and exacerbates encephalitis and neurological disease.

BALB/c mice were immunized subcutaneously with soluble Neospora caninum tachyzoite antigen (NSO) entrapped in nonionic surfactant vesicles (NISVs) or administered with Freund's complete adjuvant (FCA). Following virulent parasite challenge, groups of mice immunized with NSO and either NISVs or FCA had clinical neurological disease and increased numbers of brain lesions compared to groups of mice inoculated with FCA, NISVs, or phosphate-buffered saline (PBS) alone. Increased numbers of brain lesions were statistically significant only between mice immunized with NISV-NSO and NISV- or PBS-treated mice. Following parasite challenge, brain inflammatory infiltrates in all experimental and control groups of mice were relatively similar and consisted of compact infiltrates of macrophages admixed with various numbers of lymphoid cells. Increased brain lesions in NSO-immunized mice were associated with increased antigen-specific interleukin 4 (IL-4) secretion and increased IL-4:gamma interferon secretion ratios from splenocytes in vitro and increased antigen-specific immunoglobulin G1 (IgG1):IgG2a ratios in vivo. Thus, immunization with whole killed N. caninum antigen and either liposoidal or Freund's adjuvant induced a type 2 immune response that was associated with worsened disease. The present studies emphasize the need to identify specific N. caninum antigens or other delivery systems that will elicit protective immune responses to neosporosis.

Characterization of allelic variation in the Babesia bovis merozoite surface antigen 1 (MSA-1) locus and identification of a cross-reactive inhibition-sensitive MSA-1 epitope.

The Babesia bovis merozoite surface antigen 1 (MSA-1), a member of the variable merozoite surface antigen (VMSA) family, is an immunodominant glycoprotein which elicits antibodies that inhibit erythrocyte invasion. While antigenic polymorphism is a general feature of vmsa genes, the molecular basis and extent of msa-1 sequence polymorphism have not been well characterized. In this study we defined the msa-1 locus in the biologically cloned Mexico Mo7 strain of B. bovis and identified the sequence differences between MSA-1 antigenically dissimilar strains. We then determined whether sequences conserved between distinct msa-1 alleles would induce cross-reactive CD4(+) T lymphocytes or inhibitory antibodies. The msa-1 locus in Mo7 contains a single msa-1 gene flanked by transcribed genes with no sequence homology to members of the VMSA gene family. Argentina B. bovis strains R1A and S2P have msa-1 genes with amino acid sequences that are 98.8% identical to each other, and antibodies against S2P MSA-1 cross-react with native R1A MSA-1. In contrast, identity between the Argentina and Mexico Mo7 msa-1 alleles is only 52%, with no continuous stretch of identity longer than 16 amino acids. Despite limited sequence conservation, antibodies against R1A MSA-1 were able to inhibit invasion of erythrocytes by Mo7 merozoites. The results indicate that inhibition-sensitive epitopes are conserved despite significant sequence divergence between Mexico and Argentina strain alleles and support a conserved functional role for polymorphic MSA-1 in erythrocyte invasion.

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.

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.

Analysis of the 16S rRNA gene of micro-organism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

The structural gene encoding the 16S rRNA of the new obligate intracellular organism presently designated WSU 86-1044T was sequenced and analysed to establish its phylogenetic relationships. The 16S rDNA sequence was most closely related to those of chlamydial species, having 84.7-85.3% sequence similarity, while it had 72.4-73.2% similarity with rickettsia-like organisms. When the sequences of the four species of chlamydiae (Chlamydophila psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila pecorum) were compared, they had > 93% sequence similarity indicating that WSU 86-1044T was not close enough to be in the same family as current Chlamydiaceae members. However, based on the 84.7-85.3% 16S rDNA sequence similarity of WSU 86-1044T and other previously described characteristics, WSU 86-1044T belongs to a novel family within the order Chlamydiales; hence, the proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov.

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle.

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.

Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.

Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion, bovine IL12 and IL4 expressed by recombinant vaccinia virus in vitro clearly exhibited type 1-type 2 modulating properties.

Bovine CD4(+) T-lymphocyte clones specific for rhoptry-associated protein 1 of Babesia bigemina stimulate enhanced immunoglobulin G1 (IgG1) and IgG2 synthesis.

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-gamma)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) of Babesia bigemina induces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-gamma and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4(+) T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-gamma and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4(+) Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasite Fasciola hepatica that expressed relatively more IL-4 than the B. bigemina-specific Th cells were similarly assayed. B. bigemina RAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific for F. hepatica enhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-gamma by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response in B. bigemina RAP-1 immune cattle and a biased IgG1 response in F. hepatica-immune cattle.

Persistence of Anaplasma ovis infection and conservation of the msp-2 and msp-3 multigene families within the genus Anaplasma.

Goats which have recovered from acute Anaplasma ovis infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of A. ovis 17 to 21 months following experimental infection was demonstrated by PCR detection of the msp-5 gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10(2) organisms/ml to peaks of 10(6) organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for A. marginale-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The msp-2 and msp-3 multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent A. marginale rickettsemia, were identified in the A. ovis genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.

CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.

The rhoptry-associated protein 1 (RAP-1) expressed by all babesial parasites is encoded by tandemly arranged genes separated by discrete intergenic (IG) regions. We hypothesize that these IG regions regulate rap-1 gene expression. In Babesia bovis two identical rap-1 gene copies are separated by a 1.0-kb noncoding region which is also exactly conserved 5' to the rap-1 gene 1. In contrast, the complex B. bigemina rap-1 locus contains at least 5 polymorphic rap-1a genes separated by uncharacterized 3.38-kb regions. A genomic clone encoding the 3' sequence of rap-1 gene copy 1, the 1 kb IG region, and the 5' sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biological clone of B. bovis and sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating two B. bigemina rap-1a genes, revealing the presence of two different IG regions denominated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identified rap-1b orf. Sequence analysis and comparison among babesial rap-1 IG regions from B. bovis, B. bigemina, B. canis, and B. ovis revealed conservation of at least three putative regulatory boxes consistently positioned 5' of the start of the rap-1 orfs. To determine whether rap-1 IG regions contained a functional promoter, the entire 1-kb IG region from B. bovis was cloned into pCAT, a promoterless plasmid containing the cat gene. The IG region in the 5' --> 3' orientation strongly promoted transcription in vitro by homologous B. bovis RNA polymerases. The presence of conserved regions 5' to each rap-1 gene copy and among other babesial rap-1 IG regions and the in vitro promoter function in the 5' --> 3' orientation support a role for the IG region in rap-1 gene regulation.