Coronary artery calcium is associated with lpa gene variant RS7765803-C in Mexican mestizo population The GEA Project

ABSTRACT Background: Lipoprotein(a) [Lp(a)] levels are genetically determined; high levels are a risk factor for coronary disease, although their association with coronary artery calcium (CAC) is controversial. Objective: The objective of the study was to assess the association of LPA gene polymorphisms with CAC in a Mexican Mestizo population. Methods: We included 1594 subjects 35-70 years old. Six polymorphisms of the LPA gene were analyzed. CAC score was determined by tomography and Lp(a) serum levels by immunonephelometry. The association of LPA polymorphism with CAC and Lp(a) was evaluated by logistic regression. Results: The prevalence of Lp(a) ≥30 mg/dL was 10%, and of CAC >0 was 26.9%. Three polymorphisms were associated with high Lp(a) levels: rs10455872-G (p = 0.013), rs6907156-T (p = 0.021), and rs7765803-C (p = 0.001). Homozygotes (CC) for the rs7765803 variant compared with the G allele (CG + GG) carriers had higher Lp(a) levels (8.9 [3.3-23.9] vs. 4.9 [2.3-11.2] mg/dL; p = 0.015) and higher prevalence of CAC >0 (36.5% vs. 26.3%, p = 0.045) and were associated with CAC > 0 (odds ratio = 1.7, 95% confidence interval: 1.06-2.7; p < 0.026). The other polymorphisms were not associated with CAC. Conclusions: This is the first study to demonstrate in a Mexican Mestizo population that carriers of the rs7765803-C allele of LPA gene have 2.6 times greater risk for high Lp(a) values and 1.7 times higher risk for coronary artery disease.

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.

p.Q192R SNP of PON1 seems not to be Associated with Carotid Atherosclerosis Risk Factors in an Asymptomatic and Normolipidemic Brazilian Population Sample / O SNP p.Q192R da PON1 não Parece Associar-se a Fatores de Risco para a Aterosclerose Carotídea em Amostra de População Brasileira Normolipidêmica e Assintomática

Evidences suggest that paraoxonase 1 (PON1) confers important antioxidant and anti-inflammatory properties when associated with high-density lipoprotein (HDL).

Distrofia muscular facioescapulohumeral en Chile: presentación de serie en hospital de referencia terciario / Facioscapulohumeral muscular dystrophy: Report of seven patients

Background: Facioscapulohumeral muscular dystrophy is the third most common muscular dystrophy with an estimated prevalence of 1 per 20.000 and a normal life expectancy in the majority of patients. However, approximately 15% of patients become wheelchair bound in the course of their life. It is a hereditary autosomal dominant disease with high (95%) penetrance by the age of 20, but with variable degree of phenotypic expression even in the same family group. Symptoms frequently start in the second decade of life, with facial and scapular weakness. Aim: To report the clinical features of seven patients with the disease, seen at a public hospital. Material and Methods: Analysis of seven patients with genetic study seen in a public Hospital in Santiago. Results: The age of patients fluctuated from 18 to 61 years and four were females. The mean age at onset of symptoms was 29 years and four had a family history of the disease. The usual presenting complaint was arm or shoulder asymmetric weakness. Four patients had bone pain. Facial involvement was present in four. A genetic study was done in five patients, the other two patients were relatives, confirming the contraction or lower number of repetitions in D4Z4 region. After 12 years of follow up only 2 patients older than 60 years cannot work and one female patients is in a semi dependent state at the age of 30. Conclusions: The clinical workup in the diagnosis and the timely indication of genetic studies are highlighted, to avoid unnecessary and invasive procedures. The variability in the phenotypic expression in a similar genetic defect is discussed and the genetic or epigenetic mechanisms of this muscular dystrophy are described.

Genotipificación y evaluación de la dinámica de infección de un aislamiento colombiano de Leptospira santarosai en el modelo experimental en hámster / Genotyping and evaluation of infection dynamics in a Colombian isolate of Leptospira santarosai in hamster as an experimental model

Introducción. Es necesario desarrollar modelos de estudio de la leptospirosis. Objetivo. Genotipificar un aislamiento de Leptospira proveniente de una persona con síndrome de Weil y evaluar, con el modelo experimental en Mesocricetus auratus , su dinámica de infección. Materiales y métodos. Se hizo la genotipificación por análisis de las secuencias génicas rrs 16S y lipL32 . Se determinó la dosis letal media en hámster inoculada por vía intraperitoneal. Se identificaron los patrones de química clínica, la duración de la leptospiremia, la leptospiruria y la histopatología, comparados con el mismo modelo inoculado con la cepa de Leptospira interrogans (Fiocruz L1-130). Resultados. Mediante análisis molecular se determinó que el aislamiento correspondía a la especie patógena Leptospira santarosai . La bacteria se recuperó a partir de tejido de riñón y de pulmón, y se detectó por medio de PCR lipL32 en el tercer día después de la infección. La proteína C reactiva aumentó en el quinto día después de la infección (3,25 mg/dl; valor normal: 0,3 mg/dl) con una disminución en el día 18 (2,60 mg/dl; valor normal: 0,8 mg/dl). Los biomarcadores de urea mostraron alteraciones indicativas de falla renal aguda (día 5 después de la infección: 49,01 mg/dl y día 18: 53,71 mg/dl). La histopatología mostró neumonía intersticial con diferentes grados de hemorragia, así como nefritis intersticial. Conclusión. Se identificó la presencia de la especie L. santarosai con capacidad patógena comparable con la cepa Fiocruz L1-130 de L. interrogans , de reconocida virulencia y tropismo pulmonar, en cuanto a los aspectos histopatológicos de tropismo a pulmón y riñón. Nunca antes se había evaluado en un modelo experimental un aislamiento de origen local bajo estos criterios biológicos.

Introduction: Is necessary to develop models for the study of leptospirosis. Objective: To genotype a Colombian strain of Leptospira isolated from a human with Weil´s syndrome and to evaluate its infection dynamics in the hamster experimental model. Materials and methods: Genotyping was performed by amplification and sequence analysis of the rrs 16S and lipL32 genes. The median lethal dose was determined in intraperitoneally inoculated hamsters. The patterns of clinical chemistry, the duration of leptospiremia, leptospiruria and pathological findings were studied and compared in the same animal model infected with L. interrogans (Fiocruz L1-130). Results: Molecular typing revealed that the isolate corresponded to the pathogenic species L. santarosai, which was recovered from hamsters´ kidneys and lungs and detected by lipL32 PCR from day 3 post-infection in these organs. There was a marked increase of C-reactive protein in animals at day 5 post-infection (3.25 mg/dl; normal value: 0.3 mg/dl) with decreases by day 18 (2.60 mg/dl: normal value: 0.8 mg/dl). Biomarkers of urea showed changes consistent with possible renal acute failure (day 5 post-infection: 49.01 mg/dl and day 18 post-infection: 53.71 mg/dl). Histopathological changes included interstitial pneumonia with varying degrees of hemorrhage and interstitial nephritis. Conclusion: The pathogenic species L. santarosai was identified in Colombia. Its pathogenicity as determined by tropism to lung and kidney was comparable to that of L. interrogans Fiocruz L1-130, well known for its virulence and pulmonar tropism. The biological aspects studied here had never before been evaluated in an autochthonous isolate.

The involvement of multiple thrombogenic and atherogenic markers in premature coronary artery disease

OBJECTIVE: To examine the association of atherogenic and thrombogenic markers and lymphotoxin-alfa gene mutations with the risk of premature coronary disease. METHODS: This cross-sectional, case-control, age-adjusted study was conducted in 336 patients with premature coronary disease (<50 years old) and 189 healthy controls. The control subjects had normal clinical, resting, and exercise stress electrocardiographic assessments. The coronary disease group patients had either angiographically documented disease (>50% luminal reduction) or a previous myocardial infarction. The laboratory data evaluated included thrombogenic factors (fibrinogen, protein C, protein S, and antithrombin III), atherogenic factors (glucose and lipid profiles, lipoprotein(a), and apolipoproteins AI and B), and lymphotoxin-alfa mutations. Genetic variability of lymphotoxin-alfa was determined by polymerase chain reaction analysis. RESULTS: Coronary disease patients exhibited lower concentrations of HDL-cholesterol and higher levels of glucose, lipoprotein(a), and protein S. The frequencies of AA, AG, and GG lymphotoxin-alfa mutation genotypes were 55.0%, 37.6%, and 7.4% for controls and 42.7%, 46.0%, and 11.3% for coronary disease patients (p = 0.02), respectively. Smoking, dyslipidemia, family history, and lipoprotein(a) and lymphotoxin-alfa mutations in men were independent variables associated with coronary disease. The area under the curve (C-statistic) increased from 0.779 to 0.802 (p<0.05) with the inclusion of lipoprotein(a) and lymphotoxin-alfa mutations in the set of conventional risk factors. CONCLUSIONS: The inclusion of lipoprotein(a) and lymphotoxin-alfa mutations in the set of conventional risk factors showed an additive but small increase in the risk prediction of premature coronary disease. .

Effect of garlic extract on some serum biochemical parameters and expression of npc1l1, abca1, abcg5 and abcg8 genes in the intestine of hypercholesterolemic mice.

Some compounds in the garlic inhibit cholesterol synthesis, resulting in lowering of serum cholesterol and triglycerides and increase in HDL level. However, the mechanism of this specific effect is not fully understood. In the small intestine, ATP-binding cassette transporters G5, G8 and A1 (ABCG5, ABCG8 and ABCA1), as well as Niemann-Pick C1 like 1 (NPC1L1) protein have important roles in cholesterol metabolism. In this study, we evaluated the beneficial effect of aqueous extract of garlic on lipid profile and also expression of npc1l1, abca1, abcg5 and abcg8 genes in the intestine of N-Marry mice fed a high cholesterol diet as a possible mechanism of garlic effect. Twenty-four mice were randomly divided into three groups: Group 1: hypercholesterolmic (received chow + 2% cholesterol + 0.5% cholic acid); Group 2: garlic (received chow + 4% (w/w) garlic extract + 2% cholesterol + 0.5% cholic acid); and Group 3: received chow only. After one month, mice were anesthetized and blood was collected from their heart. The jejunum was removed, washed with PBS and entrocytes were scraped and used for the experiments. Serum lipids were measured enzymatically and expression of mRNA levels for the above-mentioned proteins was determined by semi-quantitative RT-PCR. Garlic extract significantly reduced serum lipids (p<0.05), compared with the hypercholesterolemic group. Expression of the intestinal npc1l1 was significantly decreased (p<0.01) in the garlic group, compared with the chow group, while abcg5 (p<0.01), abcg8 (p<0.01) and abca1 (p<0.05) expressions were significantly increased. In conclusion, this study reveals a possible mechanism for the beneficial effects of the garlic in lowering serum lipids by decreasing the intestinal lipid absorption and increasing excretion of cholesterol back into the intestinal lumen.

Histopathological kidney alterations in rats naturally infected with Leptospira / Alteraciones histopatológicas en ratas naturalmente infectadas con Leptospira

Introduction: Histopathological changes by Leptospira in naturally infected rodent reservoirs have been poorly described. Objective: The aim of the current study is to describe renal histopathology associated with leptospirosis infection of naturally infected rodents captured in the urban area of the city of Medellin, Colombia. Materials and methods: We performed hematoxilin-eosin (H-E) on kidney samples collected from 254 captured rodents. The positive samples were processed by Warthin Starry (W-S) staining and PCR- LipL 32. Results: Fifty one rodent kidneys showed H-E histopathological changes that consisted of inflammatory infiltrate with lympho-plasmocitary cells and histiocytes. We performed W-S staining and PCR- LipL 32 to 67 kidney samples, including the 51 that had shown detectable changes by H-E and 16 (8%) of 203 rodents with negative results. Eight of the samples that tested positive for H-E (15.7%) were also positive for W-S staining. All negative for H-E were also negative for W-S staining. Of the W-S positive samples also tested for culture only three tested positive for both. Additionally, 47 (92.1%) samples positive for H-E were positive for PCR; while eleven of the 16 (68.8%) negative for H-E were positive for PCR. The samples positive for PCR were subsequently tested for culture and 11 (23.4%) were positive. Seven samples were positive for PCR and W-S and three were positive for PCR, W-S and culture. All of the PCR- LipL 32 fragments were sequenced and showed specific amplicons for L. interrogans . Conclusions: The Leptospira infection was confirmed in all of the animals tested. The only histological kidney lesion attributable to leptospiral infection in the reservoir was interstitial nephritis.

Introducción. Los hallazgos histopatológicos ocasionados por Leptospira spp. han sido poco estudiados en poblaciones de roedores naturalmente infectados. Objetivo. Describir la histopatología renal asociada con las infecciones naturalmente adquiridas en un grupo de roedores capturados en el área urbana de Medellín, Colombia. Materiales y métodos. Se llevaron a cabo coloraciones de hematoxilina y eosina de los riñones de 254 roedores recolectados en el área de estudio. Las muestras positivas se procesaron con la coloración de Warthin-Starry y mediante reacción en cadena de la polimerasa (PCR)-LipL32. Results. Se observaron cambios histopatológicos con hematoxilina y eosina en 51 riñones de roedores, que consistieron en infiltrado inflamatorio con linfoplasmocitos e histiocitos. Se utilizó coloración de Warthin-Starry y PCR-LipL32 en 67 muestras de riñón que incluyeron las 51 muestras que tuvieron cambios detectables por hematoxilina y eosina y 16 de 203 (8 %) muestras con resultados negativos. Ocho de las muestras positivas por hematoxilina y eosina (15,7 %) también fueron positivas por la coloración de Warthin-Starry. Las muestras negativas por hematoxilina y eosina (8 %) también fueron negativas con la coloración de Warthin-Starry. Tres de las ocho muestras positivas por esta última, también lo fueron por cultivo. Además, 47 (92,1 %) muestras positivas por hematoxilina y eosina fueron positivas por PCR. Del grupo de 16 negativos por hematoxilina y eosina, 11 (68,8 %) fueron positivos por PCR. De las muestras positivas por PCR, 11 también lo fueron por cultivo (23,4 %). Siete muestras fueron positivas por PCR y Warthin-Starry y tres lo fueron por PCR, Warthin-Starry y cultivo. Todos los fragmentos de la PCR-LipL32 fueron secuenciados y mostraron secuencias específicas de L. interrogans . Conclusiones. Se confirmó la infección por Leptospira y la única lesión presente en el reservorio atribuible fue la nefritis intersticial.

La respuesta terapéutica a ezetimiba en ratones C57BL/6 es mediada por cambios en la expresión de NPC1L1, ABCG5 y ABCG8 en el enterocito / Therapeutic response to ezetimibe in C57BL/6 mice is mediated by changes in NPC1L1, ABCG5 and ABCG8 expression in the enterocyte

Las proteínas NPC1L1, ABCG5 y ABCG8 participan en la absorción intestinal de colesterol. Ezetimiba inhibe este proceso bloqueando a NPC1L1, sin embargo, su efecto sobre ABCG5 y ABCG8 aún no está claro. Así, el objetivo del presente trabajo fue evaluar en ratones C57BL/6 con hipercolesterolemia inducida por dieta y tratados con ezetimiba, la expresión de NPC1L1, ABCG5 y ABCG8 mediante PCR en tiempo real y Western blot, en 3 grupos de animales: 1, dieta hipercolesterolémica D12336; 2, dieta D12336 más 5 mg/kg/día de ezetimiba; 3, dieta control. El nivel sérico de colesterol total fue significativamente diferente entre los grupos estudiados (control: 1,85 +/- 0,49 mmol/L; dieta D12336: 3,11 +/- 0,73 mmol/L; ezetimiba: 2,11 +/- 0,50 mmol/L, P = 0,001). La expresión génica de NPC1L1 aumentó 5,4 veces en el grupo que recibió la dieta D12336 (P = 0,003). Por otro lado, la expresión génica de ABCG5 y ABCG8 no fue diferente en el grupo con hipercolesterolemia (P = 0,239 y P = 0,201, respectivamente). Después del tratamiento con ezetimiba, la expresión génica de ABCG5 se incrementó 15,6 veces (P = 0.038). No hubo diferencias significativas en la expresión génica de NPC1L1 (P = 0,134) y ABCG8 (P = 0,067). En relación a la expresión proteica, la dieta D12336 incrementó los niveles de expresión de NPC1L1 (P = 0,022) y ABCG5 (P = 0,008); el tratamiento con ezetimiba incrementó los niveles de NPC1L1 (P = 0,048) y redujo los niveles de ABCG5 (P = 0,036) y ABCG8 (P = 0,016). En conclusión, nuestros resultados sugieren que tanto la dieta hipercolesterolémica como el tratamiento con ezetimiba, en un modelo experimental, afectan los niveles de expresión de NPC1L1, ABCG5 y ABCG8, sugiriendo que ABCG5 y ABCG8 están involucrados en la respuesta hipolipemiante a este fármaco. No obstante, el mecanismo mediante el cual se explica esta interacción requiere de un futuro estudio.

Proteins NPC1L1, ABCG5 and ABCG8 are involved in the intestinal absorption of cholesterol. Ezetimibe inhibits this process by blocking NPC1L1, however, its effect on ABCG5 and ABCG8 is not yet clear. Thus, the objective of this study was to evaluate in C57BL / 6 mice with diet-induced hypercholesterolemia treated with ezetimibe, the expression of NPC1L1, ABCG5 and ABCG8 by real time PCR and Western blot, in 3 groups of animals: 1, diet hypercholesterolemic D12336, 2, D12336 diet plus 5 mg/kg/ day of ezetimibe, 3, diet control. The serum level of total cholesterol was significantly different between groups (control: 1.85 +/- 0.49 mmol / L; diet D12336: 3.11 +/- 0.73 mmol / L; ezetimibe: 2.11 +/- 0.50 mmol / L, P = 0.001). NPC1L1 gene expression increased 5.4-fold in the group receiving the diet D12336 (P = 0.003). Furthermore, the gene expression of ABCG5 and ABCG8 was not different in the group with hypercholesterolemia (P = 0.239 and P = 0.201, respectively). After treatment with ezetimibe, ABCG5 gene expression was increased 15.6 times (P = 0.038). No significant differences in gene expression of NPC1L1 (P = 0.134) and ABCG8 (P = 0.067). Regarding protein expression, the D12336 diet increased the levels of expression of NPC1L1 (P = 0.022) and ABCG5 (P = 0.008), treatment with ezetimibe increased the levels of NPC1L1 (P = 0.048) and reduced levels of ABCG5 (P = 0.036) and ABCG8 (P = 0.016). In conclusion, our results suggest that both hypercholesterolemic diet as treatment with ezetimibe, in an experimental model, affect the expression levels of NPC1L1, ABCG5 and ABCG8, suggesting that ABCG5 and ABCG8 are involved in lipid-lowering response to this drug. However, the mechanism by which this interaction is explained requires further study.

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.

Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2

Outer membrane proteins of Pasteurella (P.) multocida have been known to be protective immunogens. Pasteurella lipoprotein E (PlpE) has been reported to be an important cross reactive outer membrane protein in P. multocida. The gene encoding the PlpE of P. multocida serotypes A: 3, B: 2 and D: 1 was amplified from the genomic DNA. The amplified products were cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clones revealed a single open reading frame of 1,011 bp, 1,008 bp and 1,017 bp encoding a protein with a calculated molecular mass of 37.829 kDa, 37.389 kDa and 37.965 kDa for serotypes A: 3, B: 2 and D: 1 respectively. The comparison of the plpE sequence in different capsular types revealed a high degree (>90%) of homology. Furthermore, the plpE gene of Haemorhhagic septicaemia causing serotype (B: 2) was expressed in E. coli and recombinant PlpE was strongly immunostained by antiserum against whole cell antigen, indicating that the protein is expressed in vivo.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

Genetic Variability of Antigen B among Echinococcus granulosus Egyptian Isolates

Genetic polymorphisms of encoding antigen B2 gene (AgB2) in Echinococcus granulosus were studied using PCR-RFLP and DNA sequencing among 20 Egyptian isolates. Five isolates from different host origins (humans, camels, pigs, and sheep) were collected and used. All examined isolates of each host group gave very similar patterns of PCR-RFLP after restriction enzyme digestion with AluI, with the gene size of approximately 140 bp and 240 bp for sheep and human isolates, and approximately 150 bp and 250 bp for pig and camel isolates. No digestion pattern was obtained after incubation of all studied isolates with EcoRI. These results reveal high intra-group homogeneity. DNA sequence analysis highlighted that human infecting strain showed 100% identity with respect to sheep infecting isolate, 96% and 99% with pig and camel infecting isolates, respectively.

Detection of pathogenic leptospires in animals by PCR based on lipL21 and lipL32 genes.

Efficacy of primers capable of amplifying conserved outer membrane protein (OMP) genes i.e., lipL21 and lipL32 of Leptospira strains was tested for rapid and early diagnosis of the leptospirosis using a polymerase chain reaction (PCR). These OMP genes were found to be conserved in various leptospiral serovars viz., Canicola, Pomona, Icterohaemorrhagiae, Pyrogenes, Sejroe, Grippotyphosa, Ballum and Tarassovi as PCR products of 561 bp and 756 bp were obtained by PCR employing lipL21 and lipL32 based primers, respectively, in all these serovars. Absence of such amplicons in DNA extracted from Pasteurella, Campylobacter and Brucella confirmed the specificity of the primers. Serum and tissue samples collected from cattle, buffaloes and experimentally infected guinea pigs and calves were subjected to PCR using above primers as well as conventionally used primers G1/G2. All the sera and tissue samples, whether field samples or collected from experimentally infected animals, found positive for G1/G2 specific PCR were also positive for lipL21 and lipL32 specific PCR. The present study indicated that lipL21 and lipL32 based primers could be used for PCR based diagnosis of leptospirosis. Since G1/G2 primers are known not to amplify the DNA of Grippotyphosa, the use of primers employed in the present study could have an additional advantage in detection of cases of the disease.

Searching for secreted proteins of Mycobacterium leprae.

In mycobacteria secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in Mycobacterium tuberculosis culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied for Mycobacterium leprae since secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of M. tuberculosis were compared with sequences of M. leprae. Genes for homologues of the 85a, 85b, 85c, mpt32 (apa), mpt51, erp, mtc28, mtb12, Rv3354 and Rv0526 genes were identified. All of these and six genes of the mcel operon contain signal sequences for secretion in M. leprae as well. In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organization of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis, the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.