Detection of Pathogenic Leptospira in Rats and Phylogenetic Analysis Using Outer Membrane Lipoprotein Lipl32 Gene at Two Major Public Markets

@#Introduction: Limited studies have been documented on the presence of pathogenic Leptospira in public markets serving the community in sub-districts of Selangor. The aim of this study was to detect the presence of pathogenic Leptospira in rats using a gene encoding an outer membrane lipoprotein LipL32. Methods: Polymerase chain reaction (PCR) was performed using LipL32 primers on sixty kidney samples of rats trapped at two locations of study; Pasar Borong Selangor in Seri Kembangan and Pasar Basah Bandar Baru Bangi in Bangi. Results: Out of 60 samples analysed, 36.7% were positive for the presence of LipL32. All positive samples highly matched (>94%) nucleotide sequence for LipL32 of pathogenic Leptospira and related to the pathogens through phylogenetic analysis. Conclusion: The detection of LipL32 indicates the potential presence of pathogenic Leptospira species at public markets. Although only 60 rats were successfully trapped, the rats are mobile and might further transmit the pathogenic organisms to other areas.

Evaluation of an in-house LipL32 polymerase chain reaction for diagnosis of leptospirosis and its correlation with various serological diagnostic techniques

Background: Leptospirosis is a zoonotic disease of ubiquitous distribution. During rainy seasons, in spring and summer and also during harvest times, the risk of leptospirosis increases as there are chances of frequent contact with infected rat population which is common in Karnataka as farming is a main source of income to the people here. There is a paucity of data regarding the prevalent serovars from Karnataka. This study was undertaken as an attempt to compare a battery of tools such as immunochromatographic test (ICT), microscopic agglutination test (MAT), immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) and in-house polymerase chain reaction (PCR) to detect leptospirosis. Settings and Design: This study using consecutive sampling technique was conducted in a tertiary care centre, Mysore, Karnataka. Subjects and Methods: Samples from 783 suspected cases of leptospirosis in and around Mysore between April 2013 and April 2016 were processed. Samples from 783 patients suspected of leptospirosis were subjected to ICT, IgM ELISA, MAT and in-house PCR. Statistical Analysis Used: The statistical analysis was carried out using SPSS software version. Results: Among 783 samples tested, only 14 (1.7%) were positive by ICT, 341 (44%) were positive by IgM ELISA, 368 (47%) were positive by MAT and 393 (50.2%) were positive by in-house PCR. Conclusions: Mysore can be considered endemic for leptospirosis. The in-house PCR based on LipL32 gene proved to be useful in the early diagnosis of leptospirosis.

Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples

Abstract A modified TaqMan real-time polymerase chain reaction targeting a 138 bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy dogs were tested for presence of leptospiral DNA using the TaqMan-based assay. The results were compared with those from a well-established conventional PCR targeting the 16S RNA encoding gene associated with nucleotide sequencing analysis. The overall agreement between the assays was 94.8% (confidence interval [CI] 95% 88-100%). The newly developed assay presented 91.6% (CI 95% 71.5-98.5%) relative sensitivity (22[+] lipl32 PCR/24[+] 16S RNA and sequencing), 100% (CI 95% 96.3-100%) relative specificity and 98.7% accuracy (CI 95% 94.8-100%). The lipl32 assay was able to detect and quantify at least 10 genome equivalents/reaction. DNA extracted from 17 pathogenic Leptospira spp., 8 intermediate/saprophytic strains and 21 different pathogenic microorganisms were also tested using the lipl32 assay, resulting in amplification exclusively for pathogenic leptospiral strains. The results also demonstrated high intra and inter-assay reproducibility (coefficient of variation 1.50 and 1.12, respectively), thereby qualifying the newly developed assay as a highly sensitive, specific and reliable diagnostic tool for leptospiral infection in dogs using urine specimens.

Recombinant LipL32 Protein Developed Using a Synthetic Gene Detects Leptospiraspecific Antibodies in Human Serum Samples

Background: Synthetic biology is emerging as a viable alternative for the production of recombinant antigens for diagnostic applications. It offers a safe alternative for the synthesis of antigenic principles derived from organisms that pose a high biological risk. Methods: Here, we describe an enzyme-linked immunosorbent assay (ELISA) using the synthetic recombinant LipL32 (rLipL32) protein expressed in Escherichia coli for the detection of Leptospira-specific antibodies in human serum samples. The rLipL32-based ELISA was compared with a microscopic agglutination test (MAT), which is currently used as the gold standard for the diagnosis of leptospirosis. Results: Our results showed that all the MAT-positive serum samples were positive for Leptospira-specific IgG in an ELISA, while 65% (n = 13) of these samples were also positive for Leptospira-specific IgM. In the MAT-negative serum samples, 80% and 55% of the samples were detected as negative by an ELISA for Leptospira-specific IgM and IgG, respectively. Conclusion: An ELISA using the synthetic rLipL32 antigen was able to distinguish Leptospira-specific IgM (sensitivity 65% and specificity 80%) and IgG (sensitivity 100% and specificity 55%) in human serum samples and has the potential to serve as a rapid diagnostic test for leptospirosis.

DETECCIÓN DE Leptospira spp. PATÓGENAS EN TEJIDO RENAL DE RATÓN CASERO (Mus musculus) EN SINCELEJO, COLOMBIA / DETECTION OF PATHOGENIC Leptospira spp IN HOUSE MOUSE (Mus musculus) KIDNEY TISSUE IN SINCELEJO, COLOMBIA

La leptospirosis es una enfermedad zoonótica de distribución mundial, con mayor frecuencia en los países tropicales. Algunos roedores, y entre ellos Mus musculus o ratón casero, son portadores crónicos asintomáticos de Leptospira spp. patógenas, debido a que alojan la bacteria en sus riñones y la diseminan al ambiente a través de la orina. El presente trabajo tuvo como objetivo detectar molecularmente Leptospira spp. patógenas en riñones extraídos de M. musculus en el municipio de Sincelejo (Sucre, Colombia). La captura de los roedores se realizó en el área urbana del municipio; se instalaron trampas tipo Sherman® intra y peridomicilio en el segundo periodo de 2010 y el primero de 2011 por un lapso de 14 y 15 horas. Durante la necropsia se retiró los riñones de los individuos para la extracción de ADN y análisis de PCR para la amplificación del segmento génico de 423pb entre las posiciones 270 y 692 de la región codificante LipL32. Se capturaron 154 individuos, de los cuales 5 fueron positivos para Leptospira spp. patógenas. El análisis de correspondencias mostró que los ratones positivos se encuentran mayoritariamente en la comuna 2, seguido por la comuna 4 del municipio. El estudio sugiere que el saneamiento básico establece ambientes propicios para que se presente el proceso epidemiológico de la bacteria, y se demuestra la circulación de Leptospira spp. patógenas en M. musculus en el municipio de Sincelejo. Por lo que se infiere que la infestación de roedores en la zona de estudio es fuente potencial de transmisión de Leptospiras patógenas para el ser humano, que es susceptible a todas las especies patógenas del género.

Leptospirosis is a zoonotic disease of worldwide distribution with more frequency in tropical countries. Some rodents and among them the Mus musculus or house mouse are asymptomatic chronic carriers of pathogenic Leptospira spp because they have them in their kidneys and spread it to the environment through the urine. This study aimed to detect Leptospira spp molecularly in kidneys removed from M. musculus in the municipality of Sincelejo (Sucre, Colombia). The capture of rodents was conducted in the urban area of the city: intra and peridomiciliar Sherman® traps were installed in the second quarter of 2010 and the first quarter of 2011 for 14 to 15 hours. During the necropsy the kidneys of individuals were removed for DNA extraction and PCR analysis for the amplification of the 423 pb gene segment between positions 270 and 692 of the coding region LipL32. A total of 154 individuals were captured, of which five were positive for pathogenic Leptospira spp. The correlation analysis showed that mice are mostly positive in commune 2, followed by commune 4 in the municipality. The study suggests that basic sanitation establishes favorable environments for the epidemiological process of the bacteria and the circulation of pathogenic Leptospira spp. in M. musculus is demonstrated in the municipality of Sincelejo. It is inferred that rodent infestation in the studied area is a potential source for the transmission of pathogenic Leptospira spp. to human beings, which is susceptible to all pathogenic species of the genus.

Diversidad molecular de candidatos vacunales en Leptospira spp / Molecular Diversity of Vaccine Candidates in Leptospira spp / Diversidade molecular de candidatos para vacina em Leptospira spp

El objetivo de este estudio fue determinar la diversidad molecular de las proteínas OmpL1, LipL32, LipL41, LigA y LigB y de los genes que las codifican mediante análisis bioinformáticos en diferentes cepas patógenas de Leptospira spp., a partir de la información disponible en las bases de datos. Se utilizaron las secuencias de aminoácidos de las proteínas OmpL1, LipL32, LipL41, LigA y LigB, así como las de los genes que las codifican en las cepas de Leptospira spp. registradas en The National Center for Biotechnology Information (NCBI). Los análisis de las proteínas y los genes se realizaron mediante los recursos Protein, Nucleotide y Gene del NCBI. La alineación de las secuencias consenso se realizó con las herramientas PSI-BLAST y BLASTn. El porcentaje de cobertura de las secuencias seleccionadas de los genes ompL1, lipL32, lipL41, ligA y ligB en cepas patógenas de Leptospira spp. es de 100% para ompL1, lipL32 y lipL41, 75% para ligA y 99% para ligB con porcentajes de identidad de 85, 98, 88, 90 y 80% respectivamente; el porcentaje de cobertura de las secuencias seleccionadas de las proteínas es de 100, 77, 99, 100 y 100% con porcentajes de identidad de 90, 99, 92, 63 y 60% respectivamente, lo cual indica que los genes y las proteínas, excepto las proteínas LigA y LigB, son bastante conservadas en los diferentes serovares patógenos de Leptospira spp. Según dichos resultados, se recomienda realizar análisis complementarios de estas proteínas con el fin de determinar si es viable su uso como candidatos vacunales.

The aim of this study was to determine the molecular diversity of OmpL1, LipL32, LipL41, LigA and LigB proteins and that of the genes that encode them using bioinformatic analysis in different pathogenic strains of Leptospira spp. based on the information available in databases. The amino acid sequences of OmpL1, LipL32, LipL41, LigA and LigB proteins were used, as well as the genes encoding them in strains of Leptospira spp. reported at The National Center for Biotechnology Information (NCBI). The analysis of proteins and genes were performed using the Protein, Nucleotide and Gene resources from the NCBI. The alignment of the consensus sequences was performed using the PSI-BLAST and BLASTn tools. The coverage percentage of the selected sequences ofthe ompL1, lipL32, lipL41, ligA and ligB genes in pathogenic strains of Leptospira spp. is 100% for ompL1, lipL32 and lipL41, 75% for ligA and 99% for ligB with identity percentages of 85, 98, 88, 90 and 80% respectively; the coverage percentage of the selected protein sequences is 100, 77, 99, 100 and 100% with identity percentages of 90, 99, 92, 63 and 60% respectively, indicating that genes and proteins, except LigA and LigB proteins, are highly conserved in various pathogenic serovars of Leptospira spp. According to these results, it is recommended that further analysis of these proteins be made in order to determine the feasibility of its use as vaccine candidates.

El objetivo de este estudo foi determinar a diversidade molecular das proteínas OmpL1, LipL32, LipL41, LigA e LigB e dos genes que as codificam mediante análises bioinformáticas em diferentes cepas patógenas de Leptospira spp. A partir da informação disponível nas bases de dados. Utilizaram-se as sequências de aminoácidos das proteínas OmpL1, LipL32, LipL41, LigA e LigB, assim como as dos genes que as codificam nas cepas de Leptospira spp. Reportadas em The National Center for Biotechnology Information (NCBI). As análises das proteínas e dos genes se realizaram mediante os recursos Protein, Nucleotide e Gene do NCBI. O alinhamento das sequências consenso se realizou com as ferramentas PSI-BLAST e BLASTn. A porcentagem de cobertura das sequências selecionadas dos genes ompL1, lipL32, lipL41, ligA e ligB em cepas patógenas de Leptospira spp. É de 100% para ompL1, lipL32 e lipL41, 75% para ligA e 99% para ligB com porcentagens de identidade de 85, 98, 88, 90 e 80% respectivamente; A porcentagem de cobertura das sequências selecionadas das proteínas é de 100, 77, 99, 100 e 100% com porcentagens de identidade de 90, 99, 92, 63 e 60% respectivamente, o que indica que os genes e as proteínas, exceto as proteínas LigA e LigB, são altamente conservadas nos diferentes serovares patogênicos de Leptospira spp. Segundo esses resultados, se recomenda realizar análises complementares destas proteínas com finalidade de determinar se é viável o seu uso como candidatos para vacina.

The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response

We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES / PCR altamente sensible para la detección de Leptospira spp. patógenas en tejidos embebidos en parafina

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

El presente estudio describe el desarrollo y aplicación de un nuevo ensayo de PCR para la detección específica de leptospiras patógenas y su comparación con un protocolo reportado previamente. Se diseñaron nuevos cebadores para la optimización y evaluación de la PCR en tejidos embebidos en parafina infectados artificialmente. La PCR se aplicó además a muestras de tejidos embebidos en parafina y se realizó la secuenciación del amplicón resultante. La PCR diseñada fue más eficiente que el protocolo reportado, permitiendo la amplificación del fragmento de ADN esperado en las muestras infectadas artificialmente y del 44% de las muestras post mortem. Se secuenciaron 10 amplicones provenientes de pacientes diferentes. La aplicabilidad de una herramienta altamente sensible y específica en la búsqueda de leptospiras patógenas en especímenes histopatológicos podría facilitar una mejor valoración de la prevalencia y la epidemiología de la leptospirosis, la que constituye un problema de salud en disímiles países.

Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay

The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera.

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Diagnosis of leptospirosis by PCR is hampered due to the presence of substances on biological fluids. Here, we report an immunomagnetic separation step prior to PCR which improved the detection of Leptospira spp. in blood and urine samples from dogs. It resulted in a significant improvement on sensitivity for diagnosis of canine leptospirosis.

Rapid Diagnosis of Leptospirosis by Multiplex PCR

Background: Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance. Methods: In this study, we developed a multiplex PCR (mPCR) assay for detecting Leptospira’s DNA. The mPCR assay detects both the 16S rRNA gene and the major outer membrane lipoprotein gene, which is known as LipL32. Representative serovars were tested from 10 species of Leptospira and 23 other species of bacteria. Results: A positive result was obtained from all leptospiral serovars. The amplification sensitivity for the multiplex assay was 21.8 pg and 1 x 103 leptospires/ml. This mPCR assay has the potential to facilitate a rapid and sensitive diagnosis for acute leptospirosis. Conclusion: The mPCR assay developed in this study can be used for the early detection of leptospirosis. The LipL32 gene could also serve as another target to aid in the efficient detection of leptospiral infection because using 2 sets of primers in mPCR increases the sensitivity and specificity of the test.

Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) -based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207) blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira) culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21%) out of 100 culture positive blood samples, three (2.8%) out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001). A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001) signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Characterization of Leptospira borgpetersenii isolates from field rats (Rattus norvegicus) by 16S rRNA and lipL32 gene sequencing

The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 percent, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.

The immune responses induced by a recombinant DNA vaccine for leptospirosis:pVAX1/LipL21-LipL32 / 中国人兽共患病学报

Two highly conserved and with abundant quantity of lipoproteins in outer membrane of pathogenic species,but not in saprophytic species of leptospira ,LipL32 and LipL21,were selected to construct the fusion gene DNA vaccine pVAX1/LipL21-LipL32,and its ability to induce immune responses in BALB/c mice inoculated with this recombinant DNA vaccine was investigated in the present study.Expression of the fusion-protein LiPL21-LiPL32 was demonstrated in HEK293 cells following transfection with the fusion gene DNA vaccine and the immune responses induced after intramuscular inoculation with this DNA vaccine in BALB/c mice was then evaluated by microscopic agglutination test (MAT),meanwhile the ELISA assay was used to detect the cytokines induced.It was demonstrated that significant level of specific antibodies agglutinating antigens of Leptospira interrogans could be detected by MAT after DNA vaccine inoculation.The production of cytokines IL-10 and TNF-β in mice inoculated with DNA vaccine pVAX1/LipL21-LipL32 was significantly increased in comparison with that of the group inoculated with pVAX1 alone.These results indicate that the recombinant DNA vaccine pVAX1/LipL21-LipL32 may be of potential value to design and develop new generation of vaccines against leptospirosis.

Construction of the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans and the immuno-reactivity of its expressed products / 中国人兽共患病学报

In the present study, the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans was constructed by using PCR with linking primers, expressed in prokaryotic expression system. And the immuno-reactivity of its expressed product was determined. Meanwhile, SDS-PAGE and BioRad agarose image analyzer system was used to examine the expression output of the target recombinant protein rLipL32/1-LipL41/2, and the immune-reactivity of this recombinant protein was identified by Western blot assay. ELISA was used to detect the level of antibodies against the recombinant proteins in sera of patients with leptospirosis. It was demonstrated that the percentages of similarity in nucleotide and the putative amino acid sequences of the fusion gene lipL32/1-lipL41/2 with the corresponding sequences previously reported were 99. 9% and 98. 9%respectively. The expression output of the target recombinant protein was approximately 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLip41/2 could recognize and combine to the recombinant fusion protein rLip32/1-Lip41/2 as well. The positive rates of antibodies in 228 leptospirosis patients with the recombinant proteins rLip32/1,rLip41/2 and rLip32/1-Lip41.2 were 97.4%, 78. 5% and 99. 1% respectively. The results of the present study leads to the conclusion that the fusion gene lipL32/1-lipL41/3 with its prokaryotic expression system is successively constructed and the expressed recombinant protein shows excellent immuno-reactivity, thus suggesting that it may be used as the antigenic candidate for the development of the leptospiral genus-specific engineering vaccine and for the preparation of diagnostic kits.

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

Foram obtidos dois hibridomas secretores de anticorpos monoclonais (MAbs) que reagem com uma lipoproteína (LipL32) da membrana externa de leptospiras patogênicas. Para a produção dos hibridomas, células do baço de camundongos BALB/c, imunizados com LipL32 recombinante (rLipL32), foram fusionadas com células SP2/O-Ag14, selecionadas em meio HAT e testadas em ELISA indireto. Um dos MAbs secretados pelos hibridomas é do isotipo IgG2b e o outro do isotipo IgM. A especificidade dos MAbs foi confirmada em ELISA indireto e immunoblotting usando rLipL32 purificada, Escherichia coli (E. coli) expressando LipL32 e sorovares patogênicos e saprófitas. Os dois MAbs reagiram com a maioria dos sorovares patogênicos e não reagiram com sorovares saprófitas. Os MAbs possuem potencial para uso em testes de diagnóstico de leptospirose.

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

Foram obtidos dois hibridomas secretores de anticorpos monoclonais (MAbs) que reagem com uma lipoproteína (LipL32) da membrana externa de leptospiras patogênicas. Para a produção dos hibridomas, células do baço de camundongos BALB/c, imunizados com LipL32 recombinante (rLipL32), foram fusionadas com células SP2/O-Ag14, selecionadas em meio HAT e testadas em ELISA indireto. Um dos MAbs secretados pelos hibridomas é do isotipo IgG2b e o outro do isotipo IgM. A especificidade dos MAbs foi confirmada em ELISA indireto e immunoblotting usando rLipL32 purificada, Escherichia coli (E. coli) expressando LipL32 e sorovares patogênicos e saprófitas. Os dois MAbs reagiram com a maioria dos sorovares patogênicos e não reagiram com sorovares saprófitas. Os MAbs possuem potencial para uso em testes de diagnóstico de leptospirose.