Methods for Purifying Datura stramonium Agglutinin and Producing Recombinant Agglutinin Protein in a Heterologous Plant Host.

Datura stramonium seeds contain at least three chitin-binding isolectins as homo- or heterodimers of A and B subunits. This lectin has been used for the detection and isolation of sugar chains with N-acetyllactosaminyl structures on highly branched N-glycans. In terms of future diagnostic use, the development of a recombinant lectin will be the most effective approach for producing homogeneous lectin preparations. This chapter presents details of the procedure used for lectin purification and also describes a method that can be used for producing active recombinant homodimeric BB-isolectin in Arabidopsis plants.

Efficacy of three consecutive therapeutic plasma exchanges in major ABO-incompatible hematopoietic stem cell transplantation.

INTRODUCTION: We retrospectively analyzed data of recipients who underwent three consecutive therapeutic plasma exchanges (TPEs) before major ABO-incompatible (ABOi) hematopoietic stem cell transplantation (HSCT) in our hospital from 2012 to 2017 and evaluated the efficacy of TPE for successful ABOi HSCT. MATERIALS AND METHODS: We investigated the efficacy of TPE in 29 recipients with major ABOi HSCT based on the following: (1) requirement of red blood cell (RBC) transfusion during 100 days, (2) erythrocyte engraftment by reticulocyte count at 3 months, and (3) erythropoiesis recovery by bone marrow examination at 1 month and 3 months after ABOi HSCT. RESULTS: IgM and IgG donor-specific isoagglutinins (DSIs) of 31 cases of TPE were significantly decreased after three consecutive TPEs (IgM median, 1:32 to 1:2, P < .0001; IgG median, 1:256 to 1:8, P < .0001). We divided a total of 31 TPEs into two groups depending on their final DSI titers after TPE (group F, DSI > 1:16; group S, DSI ≤ 1:16). RBC transfusions were required more by group F (median, 12 units) than those by group S (median, 2 units, P = .001). Relative frequencies of erythrocyte engraftment and normal erythropoiesis after ABOi HSCT showed higher tendencies in group S than those in group F. DISCUSSION: Our study demonstrated that three consecutive TPEs were effective in reducing DSI titer in major ABOi HSCT. Reduction of pretransplant DSI in recipients could decrease requirement for RBC transfusion. Three consecutive TPEs are necessary for successful erythrocyte engraftment and normal erythropoiesis in this setting.

Coccinia indica agglutinin, a 17kDa PP2 like phloem lectin: Affinity purification, primary structure and formation of self-assembled filaments.

Phloem protein-2 (PP2) is an abundant soluble protein in the sieve elements in plants. Its lectin property was reported in various species. The primary structure of a 17kDa PP2 from Coccinia indica (Coccinia indica agglutinin, CIA17), determined by mass spectrometry, shows extensive homology with PP2 super family phloem lectins. Analysis of mass spectrometric data indicated the presence of 16 potential allelic variants of CIA17 with insignificant divergence in the primary structure. The primary structure contains an intramolecular disulfide bridge between Cys-34 and Cys-51, which is conserved across various cucurbit species and hence likely to be important for carbohydrate binding. CD spectroscopic studies revealed that CIA17 is rich in antiparallel ß-sheets, similar to PP2 proteins from Cucurbita maxima and Arabidopsis thaliana. CD spectra recorded at various temperatures showed very little change in the spectral intensity and shape up to 90°C, suggesting that CIA17 is a highly thermostable protein. Atomic force microscopic studies revealed that CIA17 forms filamentous structures at higher concentrations. In light of these results, we propose that CIA17 and other PP2 proteins play a role in the plant defense against pathogens by directly binding with the chitin cell wall, and also promote wound healing by forming self-assembled filaments.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry.

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W102) in the activity of the lectin.

Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani.

BACKGROUND: Mutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani. METHODS: The effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL. RESULTS: Knowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae. Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein. CONCLUSIONS: Taken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.

NICTABA and UDA, two GlcNAc-binding lectins with unique antiviral activity profiles.

OBJECTIVES: This study aimed to assess the antiviral properties of a unique lectin (NICTABA) produced by the tobacco plant, Nicotiana tabacum. METHODS: Cellular assays were used to investigate the antiviral activity of NICTABA and Urtica dioica agglutinin (UDA). Surface plasmon resonance (SPR) studies were performed to study the sugar specificity and the interactions of both lectins with the envelope glycoproteins of HIV-1. RESULTS: The N-acetyl-d-glucosamine (GlcNAc)-binding lectins exhibited broad-spectrum activity against several families of enveloped viruses including influenza A/B, Dengue virus type 2, herpes simplex virus types 1 and 2 and HIV-1/2. The IC50 of NICTABA for various HIV-1 strains, clinical isolates and HIV-2 assessed in PBMCs ranged from 5 to 30 nM. Furthermore, NICTABA inhibited syncytium formation between persistently HIV-1-infected T cells and uninfected CD4+ T lymphocytes and prevented DC-SIGN-mediated HIV-1 transmission to CD4+ target T lymphocytes. However, unlike many other antiviral carbohydrate-binding agents (CBAs) described so far, NICTABA did not block HIV-1 capture to DC-SIGN+ cells and it did not interfere with the binding of the human monoclonal antibody 2G12 to gp120. SPR studies with HIV-1 envelope glycoproteins showed that the affinity of NICTABA for gp120 and gp41 was in the low nanomolar range. The specific binding of NICTABA to gp120 could be prevented in the presence of a GlcNAc trimer, but not in the presence of mannose trimers. NICTABA displayed no antiviral activity against non-enveloped viruses. CONCLUSIONS: Since CBAs possess a high genetic barrier for the development of viral resistance and NICTABA shows a broad antiviral activity profile, this CBA may qualify as a potential antiviral candidate with a pleiotropic mode of action aimed at targeting the entry of enveloped viruses.

A chromophore-containing agglutinin from Haliclona manglaris: purification and biochemical characterization.

A new chromophore-containing agglutinin (Haliclona manglaris agglutinin (HMA)) was isolated from the tropical sponge H. manglaris. HMA was purified by a combination of hydrophobic interaction chromatography and ion exchange chromatography. Native HMA is a heterotrimer formed by two ß-chains (15 kDa) and one α-chain (22 kDa). HMA is a glycoprotein and possesses three intrachain disulfide bonds. Hemagglutinating activity of HMA was stable at neutral pH and temperatures up to 60 °C. HMA was only inhibited by thyroglobulin. Mass spectrometry sequencing and Edman degradation revealed a unique amino acid sequence of about 30%. Moreover, HMA has an organic chromophore of 581 Da, and this characteristic seems to be important to its antioxidant activity. Interestingly, while HMA showed no toxicity against Artemia nauplii and was unable to agglutinate bacterial cells, it did show a high capacity to protect ß-carotene against oxidation. Thus, our findings suggest the putative involvement of HMA in the protection of the sponge against oxidation.

HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells.

A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.

Prokaryotic expression and bioactivity analysis of N-terminus domain of Pinellia ternata agglutinin using alkaline phosphatase signal peptide.

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a two-domain monocot mannose-binding lectin. Pta-n encoding N-terminus domain of PTA (PTA-N) was fused with Escherichia coli alkaline phosphatase signal peptide (APSP) gene by polymerase chain reaction (PCR) for secretion expression. The fused nucleotide sequence apsp-pta-n was inserted into pET-28a prokaryotic expression vector by restriction enzyme digest sites (Nco I and Xho I), and then overexpressed in E. coli BL21(DE3) cells by isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction. Expressed APSP targeted the recombinant protein APSP-PTA-N into the periplasmic space, and then APSP was recognized and automatically cleaved by the membrane-bound signal peptidase. Ni-NTA chromatography was used for the purification and about 20 mg/L purified PTA-N was obtained. The minimum agglutination concentration of PTA-N determined by mice erythrocytes was 6.33 ± 0.47 µg/ml. The carbohydrate inhibition assay was carried out to determine the carbohydrate-binding property indicating PTA-N bound to specific sugars. The in vitro anti-proliferative activity towards human tumor cell lines and anti-fungal activity against Gibberella saubinetii were also demonstrated. Nuclear staining assay was performed to demonstrate PTA-N induced cell apoptosis. The results showed that PTA-N had significant biological functions, similar to native PTA. This strategy was the first time used to express plant mannose-binding lectin proteins and the product induced human tumor cell apoptosis, suggesting its potential application in biomedicine research.

Holothuria grisea agglutinin (HGA): the first invertebrate lectin with anti-inflammatory effects.

Holothuria grisea agglutinin (HGA) is a dimeric lectin of molecular mass 228 kDa by gel filtration with monomers of 105 kDa by SDS-PAGE. The lectin is highly thermostable as it retains full activity for 1 h at 70 °C. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HGA does not require any divalent metal ions. The affinity analysis of HGA showed that only mucin was able to inhibit the hemagglutinating activity. HGA administered intravenously was tested in classical models of nociception and inflammation. HGA was able to inhibit neutrophil migration into the peritoneal cavity induced by carrageenan. This inhibitory effect was 68% at a dose of 1 mg/kg. In acetic acid-induced writhing tests, a significant antinociceptive effect was observed by treatment with HGA (0.1; 1 or 10 mg/kg) reducing constrictions by 27, 90 and 84%, respectively. In formalin tests, HGA at a dose of 10 mg/kg showed antinociceptive effect only in the inflammatory phase (phase 2). Nevertheless, in hot-plate tests, HGA did not show any nociceptive effect. In rota-rod and open-field tests, HGA did not alter the animals' behavior. The treatment with HGA 10 mg/kg presented diminished myeloperoxidase activity activity (81.6% inhibition) and raised the circulating levels of NO by 50.4% when compared with the carrageenan group. HGA has demonstrated the ability to modulate the inflammatory response in models of inflammation in vivo. HGA is the first marine invertebrate lectin that showed an anti-inflammatory effect. This finding opens a new perspective on the potential of lectins from the marine environment.

Purification, characterization, and molecular cloning of a novel antifungal lectin from the roots of Ophioglossum pedunculosum.

A novel mannan-specific lectin was isolated from the roots of a traditional Chinese herbal medicine, Ophioglossum pedunculosum through ion-exchange chromatography and gel filtration. With a molecular mass of 19,835.7 Da demonstrated by MALDI-TOF analysis, this novel agglutinin was designated as O. pedunculosum agglutinin (OPA), specifically agglutinating human O erythrocytes and rabbit erythrocytes. The hemagglutination could be strongly inhibited by mannan and thyroglobulin, the activity of which was stable in pH range of 4.0-8.0 and at temperatures below 50 °C. Chemical modification studies indicated that tryptophan and arginine residues were essential for its hemagglutinating activity. Meanwhile, it showed antifungal activities toward Sclerotium rolfsii and Fusarium graminearum. In addition, to amplify cDNA of OPA by 3'/5'-rapid amplification of cDNA ends (RACE), the N-terminal 30 amino acids sequence of OPA was determined, and degenerate primers were designed. The obtained full-length cDNA of OPA contained 885 bp with an open-reading frame of 600 bp encoding a precursor protein of 199 amino acids, while the mature protein had 170 amino acids.

[Extraction of agglutinin from Oncomelania hupensis and its haemagglutination activity].

OBJECTIVE: To explore the extraction methods of agglutinin from Oncomelania hupensis snail and study its haemagglutination activity. METHODS: Protein obtained by ammonium sulfate fractionation precipitation with 20%-100% saturation of ammonium sulfate. Its haemagglutination activity was determined by rabbit erythrocytes. The precipitation which could agglutinate rabbit erythrocytes was diluted with 2.5 mg/ml D-galactose, D-fructose, D-glucose, saccharose, maltose and lactose, respectively, and then their haemagglutination activity was tested. Snail agglutinin were incubated at different temperatures (25-90 degrees C) and assayed for agglutinating activity. The effect of pH on the haemagglutination activity was determined by using the PBS buffer at different pH values (3.0-10.0). RESULTS: Oncomelania snail agglutinin exhibited high haemagglutination activity in 20%-40% saturated ammonium sulfate pellet. Lactose and galactose could inhibit the haemagglutination activity of snail agglutinin. The agglutinin showed maximum activity at pH 7.0. In temperature range of 30-70 degrees C, the haemagglutination activity decreased with increasing temperature, and all activity lost beyond 80 degrees C. CONCLUSION: Galactose/lactose specific agglutinin exists in Oncomelania snail, its haemagglutination activity is affected by pH and temperature.

Inhibition of hepatitis C virus 3a genotype entry through Glanthus Nivalis Agglutinin.

BACKGROUND: Hepatitis C Virus (HCV) has two envelop proteins E1 and E2 which is highly glycosylated and play an important role in cell entry. Inhibition of virus at entry step is an important target to find antiviral drugs against HCV. Glanthus Nivalis Agglutinin (GNA) is a mannose binding lectin which has tendency for specific recognition and reversible binding to the sugar moieties of a wide variety of glycoproteins of enveloped viruses. RESULTS: In the present study, HCV pseudoparticles (HCVpp) for genotype 3a were produced to investigate the ability of GNA to block the HCV entry. The results demonstrated that GNA inhibit the infectivity of HCVpp and HCV infected serum in a dose-dependent manner and resulted in 50% reduction of virus at 1 ± 2 µg concentration. Molecular docking of GNA and HCV glycoproteins (E1 and E2) showed that GNA inhibit HCV entry by binding N-linked glycans. CONCLUSION: These results demonstrated that targeting the HCV glycans is a new approach to develop antiviral drugs against HCV.

Marasmius oreades agglutinin (MOA) is a chimerolectin with proteolytic activity.

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA's proteolytic activity for mushroom defense against pathogens is discussed.

[Isolation and purification of Oncomelania hupensis agglutinin and determination of its molecular weight].

OBJECTIVE: To isolate and purify agglutinin from Oncomelania hupensis snail and determine its molecular weight. METHODS: Agglutinin was preliminarily isolated from snail tissue homogenate by 0%-40% saturated ammonium sulfate, and then successively purified with Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography. Bradford assay was used to determine the protein content. The agglutination activity was determined by rabbit erythrocytes. The purity of agglutinin preparations was assessed by SDS-PAGE. The molecular weight of agglutinin subunit was determined by Sephadex G-75 gel filtration. RESULTS: The specific activity of snail tissue homogenate was 21.74 titer/mg. After ammonium sulfate precipitation, Sephadex G-75 gel filtration and Sepharose 4B affinity chromatography, the specific activity of snail agglutinin from the homogenate solution increased to 61.93 titer/mg, 75.89 titer/mg and 963.86 titer/mg, respectively. SDS-PAGE analysis indicated that snail agglutinin (M, 53,000) was purified by Sephadex G-75 gel filtration and Sepharose 4B chromatography. The molecular weight of the snail agglutinin produced by Sephadex G-75 gel filtration was Mr 78,000. CONCLUSION: Combined use of salt fractionation, gel filtration and affinity chromatography can be efficient for extraction and purification of agglutinin from Oncomelania hupensis species. The snail agglutinin is characterized as mono subunit protein with a molecular weight of Mr 78,000.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: involvement of nitric oxide.

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1, 3, 5 and 7h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production.

A novel lectin with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from dried fruiting bodies of the monkey head mushroom Hericium erinaceum.

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9-12.1 and at temperatures up to 70 degrees C, but was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC(50) of 56.1 microM and 76.5 microM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC(50) of 31.7 microM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.

Primoinfección por citomegalovirus como causa de desarrollo de crioaglutininas y crioglobulinas en una paciente sometida a trasplante renal / No disponible

No disponible

Purification and partial characterization of an agglutinin from Octopus maya serum.

A 66-kDa lectin (OmA) was purified from the serum of the Yucatan peninsula endemic octopus (Octopus maya) by a single step affinity chromatography on glutaraldehyde-fixed stroma from rat erythrocytes. OmA corresponds to 0.8% of the total circulating protein in the hemolymph; it is composed of three equal subunits of 22kDa each, and 7.4% of linked carbohydrates. The amino acids' composition indicated that agglutinin contained mainly aspartic and glutamic acids, and cysteine and methionine were identified in minor proportion. OmA agglutinates mainly rat, guinea pig, and rabbit erythrocytes, and this activity is partially inhibited by galactosamine, melobiose, galacturonic acid, mannose, and methyl alpha and beta galactosides. Hemagglutinating activity is not dependent on divalent cations, such as Ca(2+), Mg(2+), or Mn(2+). The OmA subunits showed no identity for any lectin in databases but partial identity with the type A hemocyanin from Octopus dolfleini hemolymph; the main similarities are related to tyrosinase domains and copper A and B sites that conform to the oxygen-binding site of hemocyanin.

Títulos de anticorpos aglutinantes induzidos por vacinas comerciais contra leptospirose bovina / Agglutinating antibody titers induced by commercial vaccines against bovine leptospirosis

No presente estudo, 100 fêmeas bovinas foram divididas em cinco grupos de 20 animais cada. Os grupos experimentais receberam quatro diferentes vacinas comerciais (B, C, D e E), e um grupo permaneceu como controle. Amostras foram colhidas no dia da aplicação da primeira dose e nos dias 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 e 180 pós-vacinação (PV). A triagem dos animais foi feita pela análise sorológica com 6 antígenos de leptospiras, escolhendo-se os animais não reagentes. Os títulos de anticorpos foram monitorados pela soroaglutinação microscópica (SAM) com os sorovares Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona e Wolffi. Todas as vacinas induziram, aos 3 dias PV, títulos de anticorpos aglutinantes para os sorovares Hardjo e Wolffi, que persistiram até o 150º dia PV. Os sorovares Hardjo e Wolffi induziram os maiores títulos de anticorpos aglutinantes. A vacina D, apesar de não possuir o sorovar Wolffi em sua composição foi capaz de induzir anticorpos aglutinantes contra este sorovar. Somente foram detectados anticorpos contra o sorovar Canicola nos animais vacinados com a bacterina D. A vacina que induziu os maiores títulos médios de anticorpos, considerando todos os sorovares testados foi a D.

In the investigation 100 heifers were used, divided into 5 groups of 20 animals each. The four experimental groups were vaccinated using distinct commercial polyvalent bacterines: B, C, D and E, and A group was the control. Samples were collected at days 0, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 120, 150 and 180 from the first injection of the vaccine. The selection of the animals for the experimental groups was done based on a serological screening with 6 antigens of Leptospira sp. constituted by non-reagent animals. The vaccine titers were monitored using the microscopic agglutination test (MAT) for Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Wolffi serovars. All vaccines used were capable to product agglutinins for the Hardjo and Wolffi serovars observed at 3 days after vaccination, remaining until the 150th day; those serovars induced the highest titres of agglutinins. Vaccine D, in spite of not containing the Wolffi serovar, induced the production of agglutinins to this serovar. Agglutinins to the Canicola serovar were only observed in the animals vaccinated with the D bacterine. Vaccine D induced the highest average titers of antibodies to all tested serovars.