Identification of IgG2-specific antigens in Mexican Anaplasma marginale strains.

To identify novel antigens with immunoglobulin G2 (IgG2) specificity and immunostimulant properties for bovine Th1 cells, humoral and cellular responses were studied in cattle inoculated with initial bodies from a Mexican isolate of Anaplasma marginale and challenged with a heterologous strain. Analysis of post-immunization sera by ELISA and assaying of in vitro cellular responses in peripheral blood mononuclear cells (PBMCs) cultured in the presence of protein extracts from three Anaplasma marginale strains showed positive values of optical density ELISA readings and stimulation indices in the immunized but not control cattle. Post-immunization and post-challenge sera recognized in Western blots several proteins with molecular weights ranging from 15 to 209 kDa, twelve of which were recognized by IgG2 in the three Anaplasma marginale strains. Seven of these are novel and have not been previously reported for their IgG2 specificity; three are confirmed to be major surface proteins (MSP-1a, MSP-2, and MSP-5); and the others correspond to other well-studied MSPs but were not confirmed. Partially purified fractions of protein extracts of the Mex-17 strain were tested against PBMCs cultured in vitro. One out of the seven novel proteins induced detectable lymphoproliferation (LP) of PBMCs, and interferon-gamma was detected in supernatants of PBMC cultured in the presence of two protein fractions, including the one that caused LP. It is concluded that novel antigens, particularly the 28-kDa protein, played an additional role in the protection of immunized cattle and should be considered vaccine candidates after in vivo immunization experiments are concluded.

Pancreatitis aguda severa / Severe acute pancreatitis

La fisiopatogenia de la pancreatitis aguda sigue sin ser entendida con exactitud a pesar de los avances en modelos experimentales. El tratamiento continúa siendo primariamente de soporte. La pancreatitis aguda severa constituye la forma más ominosa de presentación de dicha patología, con una mortalidad promedio del 30 por ciento, especificada como muertes tempranas (primeras dos semanas) debidas a la disfunción orgánica múltiple por liberación de mediadores inflamatorios, y tardías secundarias a infección local y de sistémica. Punto clave de la aproximación terapéutica radica en la identificación temprana de necrosis y sobreinfección, dadas sus implicaciones pronósticas. Se revisan los aspectos relacionados a la fisiopatogenia, diagnóstico, pronóstico y tratamiento; haciendo énfasis en este último tópico donde aún la evidencia es controversial en medidas como el tipo de apoyo nutricional, pancreato-colangiografía endoscópica retrógrada, somatostatina y análogos, inmunomodulación y técnicas quirúrgicas. También se propone un algoritmo de enfoque y manejo basado en Guías de Práctica Clínica. La pancreatitis aguda tiene dos presentaciones clínicas: leve o severa. La severa generalmente está asociada a la presencia de necrosis glandular, la cual incrementa la morbi-mortalidad asociada a la entidad, especialmente si existe sobreinfección. Lo anterior implica la importancia de la identificación temprana de la necrosis y las medidas terapéuticas consiguientes: cuidados intensivos y aproximaciones quirúrgicas y no quirúrgicas. La pancreatitis aguda tiene una presentación clínica y diagnóstica que generalmente incluye dolor abdominal, vómito, fiebre, taquicardia leucocitosis y elevación de las enzimas pancreáticas. Los cálculos biliares y el abuso de alcohol son referidos como las dos primeras causas en la etiología

Evolution and function of tandem repeats in the major surface protein 1a of the ehrlichial pathogen Anaplasma marginale.

The major surface protein (MSP) 1a of the ehrlichial cattle pathogen Anaplasma marginale, encoded by the single-copy gene msp1alpha, has been shown to have a neutralization-sensitive epitope and to be an adhesin for bovine erythrocytes and tick cells. msp1alpha has been found to be a stable genetic marker for the identification of geographic isolates of A. marginale throughout development in acutely and persistently infected cattle and in ticks. The molecular weight of MSP1a varies among geographic isolates of A. marginale because of a varying number of tandemly repeated peptides of 28-29 amino acids. Variation in the sequence of the tandem repeats occurs within and among isolates, and may have resulted from evolutionary pressures exerted by ligand-receptor and host-parasite interactions. These repeated sequences include markers for tick transmissibility that may be important in the identification of ehrlichial pathogens because they may influence control strategies and the design of subunit vaccines.

Anaplasma marginale inactivated vaccine: dose titration against a homologous challenge.

The present study was performed to dose-titrate an Anaplasma marginale experimental immunogen derived from partially purified initial bodies from three geographically different Mexican strains. Three five-bovine groups were inoculated twice on days zero and 21 with A. marginale initial bodies equivalent to 1.5 x 10(10) (group I), 3 x 10(10) (group II) or 6 x 10(10) (group III) infected erythrocytes mixed with STDCM adjuvant. A similar group served as non-vaccinated controls. All four groups were challenged with 1 x 10(8) infected erythrocytes from a donor cow with an increasing rickettsemia of strain MEX-15 on day 87 post-vaccination. The prepatent period was very similar for all four groups. All five non-vaccinated controls presented typical acute anaplasmosis syndrome reaching a mean of 30.9% rickettsemia and a loss of 73.4% in the packed cell volume (PCV). Two of five controls died of acute anaplasmosis. Within the vaccinated groups only one animal (group II) suffered acute disease and died. Although all the other vaccinated animals were free of clinical signs, they developed very low rickettsemias (3.2, 3.8 and 4.3%) and PCV losses of 49.9, 47.8, and 49.3% for groups I, II and III. The starting mean weight was very similar for all four groups. All animals lost weight following challenge but losses for groups I and II were lower and significantly different from group IV losses (P < or = 0.1). Although there were no significant differences among vaccinated groups, group III was more severely affected. Taken altogether, these results show a 93.3% protection against both illness and death for all groups; and 100% protection for groups I and III, and 80% for group II.

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.

Bovine anaplasmosis prevalence in northern Veracruz state, Mexico.

In order to learn more about the presence of bovine anaplasmosis in northern Veracruz state, México, paired blood and serum samples from 368 cattle were subjected to polymerase chain reaction (PCR) and complement-fixation test (CFt). The overall prevalence of Anaplasma marginale by PCR was 69.2% and seroprevalence by CFt 54.6%. Age-specific prevalence was calculated for each test. Sixty-eight percent of animals from 0 to 3 months of age already were infected (PCR-positive), compared to only 42.4% positive by serology. CFt results suggested that presence of antibody increases with age up to 18 to 36 months, decreasing thereafter. Presence of the rickettsia seems to follow the same early pattern but with a new increase in animals 36 months or older. Serology results provided a biased picture of the true prevalence of anaplasmosis. Calculated specificity and sensitivity (63.5% and 68.2%) for CFt using PCR values as true values, appear very low and unreliable. The data generated by DNA-based surveys seem more appropriate to help design and implement control or eradication programs for bovine anaplasmosis.

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.

Intraconal amphotericin B for the treatment of rhino-orbital mucormycosis.

Rhino-orbital-cerebral mucormycosis is a disease that is frequently fatal. A 39-year-old man with diabetic ketoacidosis was referred to the authors' ophthalmic service with fever, orbital apex syndrome in the right eye, lethargy, and a black eschar in the palate. He was treated with systemic and local (intraconal) amphotericin B and his ketoacidosis was controlled; exenteration was not performed. Biopsy of the palate proved mucormycosis. Eighteen months later the patient was still alive and had a blind, anatomically preserved right eye with ptosis and intact extraocular muscle function without proptosis or pain. The authors propose this alternative means of treatment to achieve higher doses of the drug at the site of infection and better cosmetic and psychological results.

CD4+ T-helper lymphocyte responses against Babesia bigemina rhoptry-associated protein I.

A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.

Bovine babesiosis: a review of recent advances.

Bovine babesiosis, caused by parasites of the genus Babesia, is one of the world's most severe tick-borne problems of cattle in temperate to tropical areas. In the Americas Babesia bovis and B. bigemina are the causative agents, with the former considered to produce the greatest economic impact. The great complexity of the relationships causal agent-vector-host has severely hindered the efforts towards the production of a safe, long-lasting, solid-protection inducing vaccine. Recent important contributions that have encouraged the study of these agents include the development of in vitro cultivation systems, procedures for the isolation of single infected-erythrocytes, density gradient-based centrifugation systems for the isolation and concentration of both infected erythrocytes and merozoites, isozyme detection and differentiation systems that help discriminate between parasite species, and development of DNA-based diagnostics and characterization protocols. Currently, the study of the cellular immune responses against these parasites is taking new endeavors in order to discern the relationship between B cells, T cells, macrophages and their products and parasites leading to the establishment of solid, long-lasting protection. In an attempt to design a rational vaccine, T cell lines and clones are being established, and phagocytosis of infected erythrocytes and their antigens studied to try to pinpoint relevant epitopes.

Molecular epidemiology of bovine anaplasmosis.

Bovine anaplasmosis presents a worldwide distribution. However, specific models for studying the epidemiology of the disease are not available. Epidemiological modeling encounters some difficulties due to a lack of culturing techniques for Anaplasma marginale, the causative agent, as well as for the lack of typing techniques to characterize strains. The chronic carrier state and the population dynamics of mechanical and biological vectors also create difficulties. In addition, conventional serology and blood smear diagnostic techniques fail to detect all chronic carriers. Fortunately the needs for the accurate typing of isolates and for detecting chronic carriers made it possible to encourage the development of new tools based on molecular epidemiology principles. A. marginale isolates can now be typed by using panels of monoclonal antibodies, and the genes coding for some major surface proteins can be expressed or analyzed by looking at the nucleotide arrangement level. In the same manner, the latest techniques for detecting A. marginale chronic infections use DNA and RNA probes, and PCR-based methods to detect A. marginale DNA from bovine blood samples with extremely low rickettsaemias. Currently all these new epidemiological tools are being incorporated to experimental models to analyze their applicability for epidemiological studies in the near future.

Cloning of in vitro propagated Babesia bigemina.

The original Babesia bigemina culture conditions were modified with regard to infected bovine erythrocyte concentration and atmospheric environment. A procedure was designed which would yield a homogeneous parasite population, beginning with a single infected erythrocyte. Calculated dilutions were made in 96-well tissue culture plates to approach one infected erythrocyte per four wells. Growth of parasites in wells was detected between 16 and 28 days after cultures were initiated. Clones were transferred to 24-well tissue culture plates for regular maintenance. Three primary clones were selected for additional recloning. The probability that the parasites detected in one well are the progeny of a single infected erythrocyte approaches 0.99 for tertiary clones.

Evaluation of a cloned Babesia bovis organism as a live immunogen.

A Babesia bovis isolate was cloned by in vitro cultivation and compared to the original cultured isolate for pathogenicity by animal inoculation. Four yearling heifers were inoculated with cloned material and 4 with the original culture. The four animals which received the cloned Babesia showed comparatively minor hematologic changes and no clinical signs. One animal died in the group that received uncloned Babesia and the mean temperature increase and mean reduction in packed cell volume (PCV) was greater in that group. The four animals receiving the cloned material were challenged 100 days following initial inoculation. All of the animals were totally immune with no significant change in temperature or decrease in PCV, whereas control (previously non-inoculated) animals developed significant (P less than 0.001) increases in temperature and severe anemia. The cloned organism appears to be a candidate live immunogen for use in endemic areas to induce protection against bovine babesiosis.

Enzymatic characterization of Babesia bovis.

Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes.

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.

Babesia bovis: purification and concentration of merozoites and infected bovine erythrocytes.

Babesia bovis merozoites, externalized by removal of infected erythrocytes from ordinary culture conditions, were completely separated from red blood cells and stroma by centrifugation in a Percoll gradient. A merozoite band formed at a point corresponding to about 1.087 g/ml specific density. Infected red blood cells were concentrated approximately fourfold to obtain greater than 49.0% parasitemia after centrifugation in Percoll. Most highly enriched fractions positioned between 1.121 and 1.123 g/ml specific density. Full parasite viability was retained.

Cryopreservation of Babesia bigemina for in vitro cultivation.

Babesia bigemina-infected RBC and merozoites were cryopreserved and used to initiate in vitro cultures in normal bovine RBC; the cryoprotectant was a final 10% polyvinylpyrrolidone in Vega y Martinez solution. A cooling rate of 20 C/min until -80 C and then rapid transfer to liquid N2 storage was satisfactory. Samples for culture initiation were rapidly thawed at 37 C, washed in Vega y Martinez solution and resuspended in complete culture media containing 10% normal bovine RBC. The optimum culture conditions to reestablish cultures were a 24-well plate (16 mm ID), 5 mm in depth, and an atmosphere of 2% to 5% O2, 5% CO2, and 93% to 90% N2.

Cloning of Babesia bovis by in vitro cultivation.

A procedure for cloning Babesia bovis was developed. The procedure was used to establish and cultivate homogeneous populations of parasites and to isolate B. bovis from carrier animals. Three different clone lines of B. bovis based on in vitro growth rates were established.