Characterization of cellular immune response in hamsters immunized with recombinant vaccines against leptospirosis based on LipL32:LemA:LigAni chimeric protein.

In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.

TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis.

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires that are exposed on the bacterial surface and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/PpAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (p < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination. KEY POINTS: • Predicted surface-exposed epitopes were identified in three leptospiral TBDRs. • An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed. • Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.

Development of ELISA Using Recombinant Proteins for the Diagnosis of Mycoplasma hyopneumoniae Infection.

In order to develop a more sensitive and reliable method for detection of serum antibodies against Mycoplasma hyopneumoniae infection in pigs, six recombinant proteins of M. hyopneumoniae (P102, P95, P46, P97 like, Lppt, and hypothetical P987) were used for the standardization of an indirect enzyme-linked immunosorbent assay (ELISA). The proteins were evaluated against 50 sera of the specific pathogen-free and 50 sera of pigs with lesions suggestive of infection. The sensitivity was 88%, 86%, 78%, 74%, 66%, and 60% for the proteins P102, P95, P46, P97 like, Lppt, and hypothetical protein P987, respectively. Moreover, the proteins were used to establish the seroprevalence in two different commercial herds (254 sera pigs from farm considered free of M. hyopneumoniae and 246 from farm with clinical signs of enzootic pneumonia and positive serology for M. hyopneumoniae) and the positive rate was 65.2% for P95, 54.6% for P102, 40.2% for P46, 37.2% for P97 like, 17.4% for the hypothetical P987, and 14% for Lppt protein. In addition, the ELISA with six recombinant proteins was compared to commercial HerdCheck kit using 118 random pig sera samples and the results showed that ELISA with recombinant proteins were more sensitive than the commercial test. These data show that the recombinant proteins P95 and P102 are potential targets to be used in diagnostic tests to detect antibodies against M. hyopneumoniae. Although more studies are necessary, this study provides insights that these recombinant proteins can be useful in epidemiological investigations and as potential biomarkers in differentiating infected animals from those vaccinated.

Tp0684, Tp0750, and Tp0792 Recombinant Proteins as Antigens for the Serodiagnosis of Syphilis.

The incidence of syphilis has increased alarmingly over the years. Its diagnosis continues to be a challenge, leading to the search for new alternative and effective methods. The objective of this study was to select and evaluate three Treponema pallidum recombinant proteins for potential use in syphilis serodiagnosis. Bioinformatics analysis was performed with three T. pallidum antigens (Tp0684, Tp0750, and Tp0792) to assess their physical, antigenic, and structural characteristics. The antigens were chemically synthesized, recombinant plasmids were expressed in Escherichia coli BL21 Star™ (DE3), and the recombinant proteins were purified by nickel affinity chromatography. The antigenicity of the recombinant proteins was evaluated by western blotting and enzyme-linked immunosorbent assay (ELISA), using the sera from patients with primary and latent syphilis. In silico analysis indicated the antigenic potential once the exposed B cell epitopes were detected in the evaluated proteins. Sera from patients with primary and latent syphilis specifically recognized rTp0684, rTp0750, and rTp0792 recombinant antigens. Moreover, the rTp0684-ELISA receiver operating characteristic (ROC) analysis showed an area under the ROC curve of 0.99, indicating high diagnostic efficacy with 97.62% specificity and 95% sensitivity. In conclusion, rTp0684 showed better potential as an antigen for the development of syphilis serodiagnosis. Thus, bioinformatic analysis can be an important tool to guide the selection of antigens for serological diagnosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01017-w.

Slow Spread of SARS-CoV-2 in Southern Brazil Over a 6-Month Period: Report on 8 Sequential Statewide Serological Surveys Including 35 611 Participants.

Objectives. To evaluate the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) over 6 months in the Brazilian State of Rio Grande do Sul (population 11.3 million), based on 8 serological surveys. Methods. In each survey, 4151 participants in round 1 and 4460 participants in round 2 were randomly sampled from all state regions. We assessed presence of antibodies against SARS-CoV-2 using a validated lateral flow point-of-care test; we adjusted figures for the time-dependent decay of antibodies. Results. The SARS-CoV-2 antibody prevalence increased from 0.03% (95% confidence interval [CI] = 0.00%, 0.34%; 1 in every 3333 individuals) in mid-April to 1.89% (95% CI = 1.36%, 2.54%; 1 in every 53 individuals) in early September. Prevalence was similar across gender and skin color categories. Older adults were less likely to be infected than younger participants. The proportion of the population who reported leaving home daily increased from 21.4% (95% CI = 20.2%, 22.7%) to 33.2% (95% CI = 31.8%, 34.5%). Conclusions. SARS-CoV-2 infection increased slowly during the first 6 months in the state, differently from what was observed in other Brazilian regions. Future survey rounds will continue to document the spread of the pandemic.

Biofilm formation of Staphylococcus aureus from milk and expression of the adhesion genes ebpS and cna at different temperatures.

This study investigated the ability of Staphylococcus aureus isolates from milk to form biofilm, through detection of adhesion genes, investigating exopolysaccharide (EPS) production and biofilm formation on polystyrene (PS) and stainless steel (SS) surfaces, and by quantifying the expression of ebpS and cna genes under different temperatures and culture media. Among the 31 isolates, the adhesion genes ebpS and cna were found in 81% and 61% of the isolates, respectively. The screening tests for phenotype revealed that 58% of the isolates were EPS producers, and 45% showed the ability to produce biofilm on PS. Nine of the 31 isolates were selected to verify their ability to form biofilm on SS, of which 3 were non-biofilm producers, 3 were poor biofilm producers, and 3 were moderate biofilm producers. However, all nine isolates produced biofilm on SS, regardless of their phenotypic profile on PS. Reverse-transcriptase quantitative PCR (RT-qPCR) revealed no variation in the expression levels of ebpS and cna genes at different temperatures, except for isolate S24 at 10 °C, for both genes tested. Moreover, RT-qPCR assays revealed that the expression levels of the adhesion genes ebpS and cna are isolate- and temperature-dependent; however, they are independent of the phenotypic biofilm-formation profile.

Monoclonal antibodies against LipL32 confer prophylactic protection against lethal leptospirosis challenge in animal model.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The commercially available vaccines are bacterins that offer limited protection, short-term effect, and serovar-specific immunity. The development of novel immunization strategies is crucial to control the infection and decrease the chances of new outbreaks. In this study, purified monoclonal antibodies (mAbs) anti-LipL32 (1D9 and mAb3) were evaluated by their capacity to bind and neutralize the pathogen improving host survival. For that, an in vitro growth inhibition assay, and in vivo passive immunization were performed in animal model. Syrian hamsters were passively immunized by three different strategies. Hamsters immunized with mAb3 6 h prior to the lethal challenge showed a significantly higher survival rate of 61.1%, and a significant reduction in tissue damage in the lungs. Cumulatively, our results showed that anti-LipL32 mAbs inhibited the growth of L. interrogans in vitro, and that passive immunization offered significant protection in animal model when administered prior to infection.

Campylobacter jejuni isolated from poultry meat in Brazil: in silico analysis and genomic features of two strains with different phenotypes of antimicrobial susceptibility.

Campylobacter jejuni is the most common bacterial cause of foodborne diarrheal disease worldwide and is among the antimicrobial resistant "priority pathogens" that pose greatest threat to public health. The genomes of two C. jejuni isolated from poultry meat sold on the retail market in Southern Brazil phenotypically characterized as multidrug-resistant (CJ100) and susceptible (CJ104) were sequenced and analyzed by bioinformatic tools. The isolates CJ100 and CJ104 showed distinct multilocus sequence types (MLST). Comparative genomic analysis revealed a large number of single nucleotide polymorphisms, rearrangements, and inversions in both genomes, in addition to virulence factors, genomic islands, prophage sequences, and insertion sequences. A circular 103-kilobase megaplasmid carrying virulence factors was identified in the genome of CJ100, in addition to resistance mechanisms to aminoglycosides, beta-lactams, macrolides, quinolones, and tetracyclines. The molecular characterization of distinct phenotypes of foodborne C. jejuni and the discovery of a novel virulence megaplasmid provide useful data for pan-genome and large-scale studies to monitor the virulent C. jejuni in poultry meat is warranted.

Complete genome sequence and in silico analysis of L. interrogans Canicola strain DU114: A virulent Brazilian isolate phylogenetically related to serovar Linhai.

Canine leptospirosis is often caused by Leptospira interrogans serovar Canicola. Infected dogs may become asymptomatic carriers of the pathogen, which leads to many public health concerns. In this work, we present the complete genome sequencing and in silico analysis from a virulent Brazilian strain of L. interrogans serovar Canicola, previously isolated from a stray dog in Sao Paulo City. Comparative genomic analysis with a reference genome allowed identification of 1031 INDELs and several arrangement variations. Out of 35,361 SNPs identified, 6780 were missense mutations and 16,114 were synonymous mutations. The Gene Ontology terms more affected by mutations were described. Interestingly, phylogenetic analyses indicated a genetic relatedness of the isolate with serovar Linhai strain 56,609. In addition, we found several virulence-related genes and main outer membrane proteins associated with pathogenesis. This genomic information about canine isolates may help to elucidate the molecular diversity and mechanisms of Leptospira spp. pathogenicity.

Presence of genes associated with adhesion, invasion, and toxin production in Campylobacter jejuni isolates and effect of temperature on their expression.

The aims of this study were to evaluate the presence of genes associated with adhesion (cadF), invasion (ciaB), and cytotoxin production (cdtA, cdtB, and cdtC) among Campylobacter jejuni isolates from a poultry slaughterhouse and to investigate the effect of different temperatures on the expression of these virulence-associated genes. A total of 88 C. jejuni isolates from cecum, liver, chicken carcasses, chilled water, and scalding water were submitted to PCR assay for detection of virulence genes. Representative isolates were selected for gene expression evaluation at 37 and 42 °C, according to their virulence gene profile and genotypic typing. All C. jejuni isolates carried the five virulence-associated genes, which play an important role in the infectious process. Differential gene expression by RT-qPCR was observed among C. jejuni isolates at 37 and 42 °C. The expression levels at 37 °C showed upregulation of the ciaB, cdtA, cdtB, and cdtC genes in five isolates, with the exception of ciaB for isolate 4. At 42 °C, upregulation was observed for ciaB and cdtC, cdtA and cdtB, and cadF in four, three, and two isolates, respectively. The C. jejuni isolates expressed the virulence genes evaluated, and the expression is gene- and isolate-dependent and varied according the temperature.

Sex Matters: Male Hamsters Are More Susceptible to Lethal Infection with Lower Doses of Pathogenic Leptospira than Female Hamsters.

A somewhat contradictory published body of evidence suggests that sex impacts severity outcomes of human leptospirosis. In this study, we used an acute animal model of disease to analyze leptospirosis in male and female hamsters infected side by side with low but increasing doses of Leptospira interrogans serovar Copenhageni. We found that male hamsters were considerably more susceptible to leptospirosis, given that only 6.3% survived infection, whereas 68.7% of the females survived the same infection doses. In contrast to the females, male hamsters had high burdens of L. interrogans in kidney and high histopathological scores after exposure to low infection doses (∼103 bacteria). In hamsters infected with higher doses of L. interrogans (∼104 bacteria), differences in pathogen burdens as well as cytokine and fibrosis transcript levels in kidney were not distinct between sexes. Our results indicate that male hamsters infected with L. interrogans are more susceptible to severe leptospirosis after exposure to lower infectious doses than females.

Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.

Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil.

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain.

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Genome of Leptospira borgpetersenii strain 4E, a highly virulent isolate obtained from Mus musculus in southern Brazil.

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.

Histological Evaluation of Bone Repair with Hydroxyapatite: A Systematic Review.

The aim of this study was to evaluate the morphological bone response in animal experiments by applying hydroxyapatite grafts in critical and non-critical size bone defects. Current report followed the guidelines established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Animal experiments were selected by assessing repair of bone defects with hydroxyapatite as bone graft and with blood clot only as control. Eight articles were identified in specialized literature and included in the meta-analysis. Statistical analysis was carried out with a random-effect model (p = 0.05). Subgroup analyses were further performed to investigate bone repair in critical and non-critical bone defects. Comprehensive analysis of bone repair outcome showed a statistically significant difference between hydroxyapatite and blood clot control (p < 0.05). Subgroup analyses showed statistically significant difference for critical bone defects (p < 0.05). No statistically significant difference was reported in non-critical bone defects (p > 0.05). Although animal studies revealed a high risk of bias and results should be interpreted with caution, the literature suggests that non-critical bone defects may heal spontaneously and without the need of a bone graft. Conversely, when critical-size defects are present, the use of hydroxyapatite bone graft improves the bone repair process.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

Recombinant BCG vaccines: molecular features and their influence in the expression of foreign genes.

Recombinant Mycobacterium bovis BCG vaccines (rBCG) were first developed in the 1990s as a means of expressing antigens from multiple pathogens. This review examines the key structural factors of recombinant M. bovis that influence the expression of the heterologous antigens and the generation of genetic and functional stability in rBCG, which are crucial for inducing strong and lasting immune responses. The fundamental aim of this paper is to provide an overview of factors that affect the expression of recombinant proteins in BCG and the generation of the immune response against the target antigens, including mycobacterial promoters, location of foreign antigens, and stability of the vectors. The reporter systems that have been employed for evaluation of these molecular features in BCG are also reviewed here.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin.

BACKGROUND: Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES: The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS: Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS: The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION: The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUND: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.