Rough vaccines in animal brucellosis: structural and genetic basis and present status

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available

Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB(core) to distinguish it from the wbk manB(O-Ag). The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness

Extended anticoagulant treatment with full- or reduced-dose apixaban in patients with cancer-associated venous thromboembolism: rationale and design of the API-CAT study

Cancer-associated thrombosis (CT) is associated with a high risk of recurrent venous thromboembolic (VTE) events that require extended anticoagulation in patients with active cancer, putting them at risk of bleeding. The aim of the API-CAT study (NCT03692065) is to assess whether a reduced-dose regimen of apixaban (2.5mg twice daily [bid]) is noninferior to a full-dose regimen of apixaban (5mg bid) for the prevention of recurrent VTE in patients with active cancer who have completed >= 6 months of anticoagulant therapy for a documented index event of proximal deep-vein thrombosis and/or pulmonary embolism. API-CAT is an international, randomized, parallel-group, double-blind, noninferiority trial with blinded adjudication of outcome events. Consecutive patients are randomized to receive apixaban 2.5 or 5mg bid for 12 months. The primary efficacy outcome is a composite of recurrent symptomatic or incidental VTE during the treatment period. The principal safety endpoint is clinically relevant bleeding, defined as a composite of major bleeding or nonmajor clinically relevant bleeding. Assuming a 12-month incidence of the primary outcome of 4% with apixaban and an upper limit of the two-sided 95% confidence interval of the hazard ratio <2.0, 1,722 patients will be randomized, assuming an up to 10% loss in total patient-years (beta=80%; alpha one-sided=0.025). This trial has the potential to demonstrate that a regimen of extended treatment for patients with CT beyond an initial 6 months, with a reduced apixaban dose, has an acceptable risk of recurrent VTE recurrence and decreases the risk of bleeding.Thrombosis and Hemostasi

Bacterial community structure associated with the addition of nitrogen and the dynamics of soluble carbon in the rhizosphere of canola (Brassica napus) grown in a Podzol

Little is known about the community structure of bacteria in the rhizosphere of canola during growth. This study was conducted to characterize the community structure of soil bacteria in relation to soluble-C dynamics in a Podzol during the growth of canola. Next generation sequencing of 16S rDNA characterized the main bacterial communities in a Podzol that was unplanted (soil alone), and planted to canola (Brassica napus) with and without N-fertilizer addition. PERMANOVA and ANOVA analysis showed that the number of operational taxonomic units and the relative abundance of bacteria were higher in canola rhizosphere than in soil alone for phyla, classes, orders and families. In soil alone and at the family level, communities of Acidobacteriaceae, Micrococcaceae, Nocardioidaceae, Intrasporangiaceae and Nitrosomonadaceae had the highest relative abundances (sequence reads ranged from 1,000 to 3,000). In the rhizosphere, communities of Sphingomonadaceae, Bacillaceae, Gaiellaceae, Micrococcaceae and Intrasporangiaceae presented the highest relative abundance (sequence reads ranged from ~8,000 to 23,000). The addition of N-fertilizer increased the relative abundance of bacteria in 5 families, decreased those in 39 and did not affect the communities in 56 other families. A two-way ANOVA showed that management, inclusive of canola and N-fertilizer, had the largest (87%) effect on the total relative abundance of soil bacteria and soluble-C content. Polynomial regression models of second order showed a close relation between the relative abundance of bacteria and soluble-C content during crop growth. We suggest that diffusional constraints depleted soluble-C in the vicinity of microbial communities to a growth limiting concentration more frequently in the soil alone than in canola rhizosphere

Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9

Yersinia enterocolitica O:9 bears a smooth lipopolysaccharide (S-LPS) of Brucella sp. O-chain A+C/Y epitopic structure and is a cause of false-positive serological reactions (FPSR) in standard tests for cattle brucellosis. Brucella S-LPS, cross-reacting S-LPSs representing several O-chain epitope combinations, Brucella core lipid A epitopes (rough LPS), Brucella abortus S-LPS-derived polysaccharide, native hapten polysaccharide, rough LPS group 3 outer membrane protein complexes, recombinant BP26, and cytosolic proteins were tested in enzyme-linked immunosorbent assays (ELISA) and precipitation tests to detect cattle brucellosis (sensitivity) and to differentiate it from FPSR (specificity). No single serological test and antigen combination showed 100% sensitivity and specificity simultaneously. Immunoprecipitation tests with native hapten polysaccharide, counterimmunoelectrophoresis with cytosolic proteins, and a chaotropic ELISA with Brucella S-LPS were 100% specific but less sensitive than the Rose Bengal test, complement fixation, and indirect ELISA with Brucella S-LPSs and native hapten or S-LPS-derived polysaccharides. A competitive ELISA with Brucella S-LPS and M84 C/Y-specific monoclonal antibody was not 100% specific and was less sensitive than other tests. ELISA with Brucella suis bv. 2 S-LPS (deficient in C epitopes), Escherichia hermannii S-LPSs [lacking the contiguous alpha-(1-2)-linked perosamine residues characteristic of Y. enterocolitica S-LPS], BP26 recombinant protein, and Brucella cytosolic fractions did not provide adequate sensitivity/specificity ratios. Although no serological test and antigen combination fully resolved the diagnosis of bovine brucellosis in the presence of FPSR, some are simple and practical alternatives to the brucellin skin test currently recommended for differential diagnosis

Rough vaccines in animal brucellosis: structural and genetic basis and present status

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available

Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB(core) to distinguish it from the wbk manB(O-Ag). The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness