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Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions.

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

Amino Acid Sequence↗

A card test for the serodiagnosis of human leptospirosis.

A macroscopic agglutination test for detecting leptospiral antibodies in human sera is described. The test utilizes a stained preparation of non-pathogenic leptospires and is performed on the Brewer diagnostic cards used for bovine brucellosis screening. The agglutination of the stained antigen is more easily observed than the current macroscopic slide test using unstained leptospiral antigens. The non-pathogenic serotype patoc is agglutinated by sera from humans infected with serotypes pomona, hardjo, ballum, and copenhageni with a sensitivity of 94% in comparison with the microscopic agglutination test.

Agglutination Tests↗

Leptospirosis in a caver returned from Sarawak, Malaysia.

This article describes a case of leptospirosis in a man who returned from caving in Sarawak, Malaysia, and includes a discussion of epidemiology, pathophysiology, diagnosis, prevention, and treatment. The patient presented with symptoms of leptospirosis, which was confirmed by microhemagglutination titers. He became infected despite taking doxycycline daily for malaria prophylaxis. Leptospirosis is an important consideration in any returned traveler with fever. The spirochete spreads from animals to humans via water. Caving in tropical endemic zones may increase exposure risk due to the combination of multiple skin abrasions with immersions. Water in caves may increase infection risk because of increased water pH. Standard prophylaxis may be inadequate in cases of high-risk exposures.

Adult↗

Comparison of polymerase chain reaction assays with bacteriologic culture, immunofluorescence, and nucleic acid hybridization for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle.

OBJECTIVE: To compare sensitivity and specificity of various polymerase chain reaction (PCR) assays for detection of Leptospira borgpetersenii serovar hardjo in bovine urine and to compare results of the optimal PCR assay with results of immunofluorescence, nucleic acid hybridization, and bacteriologic culture. ANIMALS: 6 heifers. PROCEDURE: Heifers were exposed to serovar hardjo type hardjo-bovis by conjunctival instillation of 10(6) leptospires on 3 successive days. Urine samples were collected before and after infection. Sensitivity and specificity of 5 PCR assays were compared, to determine the optimal assay for use with bovine urine samples. The optimal PCR assay was then compared with results of bacteriologic culture, nucleic acid hybridization, and immunofluorescence. RESULTS: A PCR assay with the best combination of specificity (100%) and sensitivity (91%) was selected for comparison with the other diagnostic tests. Sensitivity for nucleic acid hybridization was 55%, whereas sensitivity for bacteriologic culture and immunofluorescence was 89 to 93%. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture, PCR, and immunofluorescence were sensitive for detection of L borgpetersenii serovar hardjo type hardjo-bovis in urine specimens of cattle, but a single technique was not the most sensitive for each animal tested. Therefore, the use of 2 techniques in combination is warranted for maximal sensitivity for diagnosis.

Animals↗

[Epidemiological, clinical, biological and developmental aspects of leptospirosis: apropos of 30 cases in Aquitaine].

We have studied retrospectively 30 cases of leptospirosis observed in Aquitaine (South West France) from 1980 till 1992. This review was made in three internal and a nephrology department in Bordeaux hospital. Most cases occurred by indirect contact with infected animals or by occupational exposures. Onset was brutal with fever often associated with painful syndrome and sometimes conjunctival suffusion. Jaundice (70%), acute renal failure (67%), meningitis (50%) and hemorrhagic signs (50%) were among the major visceral manifestations. Diagnosis was always confirmed by micro-agglutination test. Leptospira ictero-hemorrhage was the predominant serogroup found. The outcome was favorable in 22 patients; reversible complications were seen in six cases (five acute renal failure with hemodialysis myocarditis and pulmonary edema in two hemodialysed patients, polyradiculoneuritis). Two patients died (acute respiratory failure and meningo-encephalitis with diffuse hemorrhagic syndrome). The characteristic of our series is the high frequency of hepatorenal syndrome due to the importance of our nephrologist recruitment. Furthermore our study confirm the vital prognostic characters of the pulmonary, renal, hemorrhagic and neurologic complications. No absolute relationship was found between the clinical and laboratory findings and the serotype of leptospira. Penicillin remains the treatment of choice and should be started as soon as possible the avoid the life threatening visceral complications.

Adolescent↗

Leptospirosis in selected wild mammals of the Florida panhandle and southwestern Georgia.

A group of 144 wild mammals, including white-tailed deer, cottontail rabbits, fox squirrels, gray squirrels, raccoons, opossums, a bobcat, and various small rodents was examined for cultural or serologic evidence of leptospiral infection. Leptospires were isolated from 1 of 25 rabbits, 1 of 27 fox squirrels, 1 of 26 gray squirrels, 4 of 18 mice and rats, 8 of 21 raccoons, 7 of 17 opossums, and a bobcat. Isolations were not made from 6 deer examined. Serotypes isolated were Leptospira interrogans, serotype grippotyphosa and L interrogans, serotype ballum. New host-serotype relationships were noticed in the following instances: bobcat: grippotyphosa, and gray squirrel: ballum. These studies further confirm the occurrence of grippotyphosa in the fox squirrel. Serologic response in these animals did not necessarily correlate with isolations, although some relationship was noticed in raccoons and opossums.

Animals↗

Natural foci of leptospirosis on the territory of the north-eastern part of the east mountain range of Azerbaijan and their characteristic.

Numerous natural foci of leptospirosis infection in which Ap. sylvaticus and M. musculus play the role of the main source, were found on the territory of the north-eastern part of Azerbaijan's east mountain range. The leading place in the aetiological structure of leptospirosis of small mammals is occupied by leptospirae of the serological group Hebdomadis. As a rule, the animals -- Leptospira-carriers -- populate damp biotopes where stable natural foci of disease are formed. Two types of natural foci were established on the given territory, i. e., the marshy-bush type and the mountain-stream type. The foci are characterized by several hosts and several serotypes of Leptospira.

Animals↗

Surveillance of some infectious diseases among aircrew personnel in Southeast Asia.

A 2-year analysis of specimens among aircrew personnel in Southeast Asia is reported. Stool specimens were examined for intestinal parasites and enteric bacteria, blood smears for blood parasites, and sera tested for transaminases (SGOT) and antibodies to Entamoeba histolytica, Toxoplasma gondii, Chikungunya and Japanese encephalitis viruses, Rickettsia tsutsugamushi, Leptospira sp. and Pseudomonas pseudomallei. One to four specimens each were obtained from 537 adult males and 20 females. There were 56 subjects with intestinal parasites as follows: Giardia lamblia (3.8%), Endolimax nana (2.6%), Entamoeba histolytica (1.4%), Entamoeba hartmanni (0.9%), Entamoeba coli (0.7%), Trichiuris trichiura (1.7%), Ascaris lumbricoides (1.4%), hookworm (0.9%), and Clonorchis sinensis (1.2%). Two individuals had malaria, Plasmodium vivax (0.6%). Pathogenic enteric bacteria were isolated from seven stool specimens; Shigella groups B and D (0.9%), Salmonella paratyphi (0.3%), and Arizona group (0.9%). Significantly elevated SGOT levels were found in 0.9% and antibodies to Japanese encephalitis virus in 1.5%, to Rickettsia tsutsugamushi in 1.2%, to Pseudomonas pseudomallei in 0.3%, to Entamoeba histolytica in 0.9% and to Toxoplasma gondii in 10.1%. No antibodies were found to Chikungunya virus or Leptospira sp.

Aerospace Medicine↗

Urea utilization by Leptospira.

One representative of each of five different pathogenic serotypes of Leptospira as well as one saprophytic strain were capable of growing on medium containing urea in place of an ammonium salt as a nitrogen source. Growth of all of the organisms tested on 1% urea was substantial, but only those that exhibited strong urease activity could grow to any appreciable extent on urea at a concentration as high as 2%. Intact urea-grown cells of the pathogenic serotypes tested (grippotyphosa and icterohaemorrhagiae) exhibited urease activity, with the level of activity of the former being considerably greater. No urease could be detected in cells of the saprophytic strain. When the pathogenic leptospires were sonicated or treated with toluene, the urease activity was greatly enhanced. When cultivated on NH(4)Cl, neither intact nor disrupted cells of any of the strains tested exhibited any urease activity. Cells of the grippotyphosa and icterohaemorrhagiae strains exhibited diauxic growth when cultivated in the presence of both NH(4)Cl and urea, whereas only monophasic growth could be detected for the saprophytic test strain. The experimental data on urea utilization and urease activity, when considered in the light of previously reported findings on leptospiral pathology, renal physiology, and the role of urease in other bacterial infections, suggests a significant role for leptospiral urease (in addition to other factors) in determining localization of the organism in the kidney and contributing to the resultant kidney pathology.

Ammonium Chloride↗

Biological activities of leptospiral lipopolysaccharide.

Lipopolysaccharide extracted with phenol-water from Leptospira interrogans serovar copenhageni strain Shibaura (L-LPS) showed various biological activities. In lethality for mice, L-LPS was active (LD 50, 3.4 mg/mouse) but about 12 times less potent than Escherichia coli LPS (E-LPS) per weight basis. L-LPS had pyrogenicity for rabbits, and the fever curves showed no evidence of the classical biphasic fever produced by E-LPS. In the bone marrow of mice, L-LPS caused hemorrhages and necrosis but less severe than those caused by E-LPS. Histopathologically, fresh hemorrhages were found in the intestine, spleen, lung and the other organs at 24 h after inoculation of L-LPS. Necrosis was also found in these organs and was particularly severe in mice inoculated with more than 2 mgL-LPS. Liver necrosis was found at 7th day after inoculation of L-LPS but not after inoculation of E-LPS. L-LPS had adjuvant activity just like E-LPS. L-LPS enhanced non-specific resistance to Salmonella infection and activated mouse peritoneal macrophages to kill these organisms. L-LPS was positive in limulus test just like E-LPS. These results demonstrated similarities of L-LPS and E-LPS. Some toxic effects of L-LPS were less than those of E-LPS, but some effects of L-LPS were more than those of E-LPS. L-LPS was antigenically active and the specificity was serogroup-associated. L-LPS was composed of carbohydrate (54%), lipid (12%), protein (5%). Arabinose, xylose and rhamnose were major sugars as detected by gas chromatography. 2-keto-deoxyoctanate (KDO) was not detectable.

Animals↗

Presence of antigen and antibodies in serum and genital discharges of heifers after experimental intrauterine inoculation with Leptospira interrogans serovar hardjo.

The excretion of Leptospira interrogans serovar hardjo in cervico-vaginal mucus (CVM) or urine and the local and systemic immune responses to the organism were monitored in eight susceptible heifers after intrauterine inoculation while six similar heifers served as controls. All the heifers were inseminated at the subsequent oestrous periods. The overall percentage pregnancy rate (the number of pregnancies divided by the total number of inseminations) was lower in the infected heifers than in the controls though not significantly (33.3 v 50.0 per cent). Leptospires were detected, in either the urine or the CVM of six of the eight infected heifers during the study period of 15 weeks, either by direct immunofluorescence or dark ground microscopy; the bacteria did not grow in culture from any of the CVM samples. The control heifers remained free from evidence of infection. In the infected heifers, mean titres of at least 1:100 in a microscopic agglutination test were maintained for one to two weeks before declining to 1:10 to 1:30, whereas in serum IgG-ELISA tests (developed by using either protein or carbohydrate antigens), antibody titres of at least 1:100 were maintained throughout the study. During oestrous periods, IgA antibodies were detected more frequently in CVM with titres which were usually higher than the titres of IgG.

Agglutination Tests↗

Changing epidemiology of human leptospirosis in New Zealand.

The objective was to describe the current epidemiology and trends in New Zealand human leptospirosis, using descriptive epidemiology of laboratory surveillance and disease notification data, 1990-8. The annual incidence of human leptospirosis in New Zealand 1990-8 was 44 per 100,000. Incidence was highest among meat processing workers (163.5/100,000), livestock farm workers (91.7), and forestry-related workers (24.1). The most commonly detected serovars were Leptospira borgpetersenii serovar (sv.) hardjo (hardjobovis) (46.1%), L. interrogans sv. pomona (24.4%) and L. borgpetersenii sv. ballum (11.9%). The annual incidence of leptospirosis declined from 5.7/100,000 in 1990-2 to 2.9/100,000 in 1996-8. Incidence of L. borgpetersenii sv. hardjo and L. interrogans sv. pomona infection declined, while incidence of L. borgpetersenii sv. ballum infection increased. The incidence of human leptospirosis in New Zealand remains high for a temperate developed country. Increasing L. borgpetersenii sv. ballum case numbers suggest changing transmission patterns via direct or indirect exposure to contaminated surface water. Targeted and evaluated disease control programmes should be renewed.

Adolescent↗

Epidemiological aspects of leptospirosis in a slum area in the city of Rio de Janeiro, Brazil. Search for leptospires and specific antibodies in rodents.

101 Rattus norvegicus and 3 Mus musculus were captured in an urban slum of Rio de Janeiro city and examined for leptospiral infection. Direct examination of urine, serology (microagglutination) and culture together gave a positivity rate of 39%. Separately direct examination and serology gave positivity rates of 13% and 36% respectively. Serogroup Icterohaemorrhagiae predominated in the serological tests. Leptospires were isolated from 24 of the 104 rodents (23%), but 4 cultures died. The remaining 20 were identified as members of the Icterohaemorrhagiae serogroup, and 5 were further identified as serovar copenhageni. Human leptospiral infection, mainly caused by Icterohaemorrhagiae, is endemic in Rio de Janeiro city, and rodents are a major source of the disease.

Agglutination Tests↗

A waterborne outbreak of leptospirosis among United States military personnel in Okinawa, Japan.

A waterborne outbreak of leptospirosis occurred among US military personnel during September 1987, on the island of Okinawa, Japan. Micro-agglutination titres for leptospiral antigen of greater than or equal to 200 were detected in hospitalized adult males averaging 22.5 years of age with symptoms compatible with leptospirosis. Epidemiological findings revealed two case clusters distinguished by time and place of exposure. The overall attack rates among recreational swimmers and combat skills training participants were 467/1000 (7/15) and 183/1000 (15/82), respectively. Swallowing of water differentiated individuals with laboratory-confirmed infection from those with no infection, while water immersion alone did not appear to result in leptospiral infection. Additionally, subnormal rainfall may have contributed to the risk of exposure associated with this outbreak.

Adult↗

Leptospiral abortions of sows: new data.

In an immunization experiment, 460 sows at 7-60 days of gestation were inoculated with a rodent-adapted live strain of Leptospira interrogans serovar tarassovi at a large swine farm previously affected by this serovar. The inoculum consisted of a haemoculture prepared from the 52nd guinea-pig passage and the dose was 2 X 10(7) leptospires per sow. Over the period from the 48th to the 73rd day after inoculation 54 out of 283 sows having been inoculated at 32-60 days of gestation, aborted or delivered weak piglets. At the same time, 177 sows inoculated at days 7-32 of gestation farrowed normal litters. Altogether 18 underdeveloped piglets 2-5 weeks old derived from naturally infected litters were killed, but leptospires were not demonstrated or cultivated from their swollen and pale kidneys. It seems that L. interrogans serovar tarassovi is able to cause foetal damage in susceptible sows at one-two months of pregnancy and that previous adaptation of this serovar to rodents does not reduce its pathogenicity for porcine foetuses.

Abortion, Veterinary↗

Production and characterization of monoclonal antibodies specific for Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno.

Murine monoclonal antibodies were produced by immunizing BALB/c mice with a killed whole-cell antigen prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis. Six of these antibodies recognized epitopes on the homologous antigen and on whole-cell antigen prepared from Leptospira interrogans serovar hardjo type hardjoprajitno. These antibodies did not cross-react with whole-cell antigens prepared from L. borgpetersenii serovar sejroe, 10 other pathogenic Leptospira serovars, or the saprophytic Leptospira biflexa serovar patoc. Three other monoclonal antibodies reacted with antigens prepared from the 2 hardjo serovars and serovar sejroe but not with antigens from the 10 other pathogenic serovars, or serovar patoc. The epitopes recognized by all of the hardjo-specific antibodies and 2 of the 3 hardjo/sejroe-specific antibodies were susceptible to sodium meta-periodate oxidation. All of the antibodies were characterized by Western blots with the hardjobovis whole-cell antigen. Each of the 9 monoclonal antibodies was inhibited from binding to the hardjobovis antigen by bovine sera which were obtained from cattle experimentally infected with hardjobovis and from field cattle, with anti-serovar hardjo microscopic agglutination test antibody titres ranging from 100 to 12800. Some of these antibodies may be suitable for incorporation into competitive enzyme immunoassays for the specific detection of antibodies to either of the hardjo serovars.

Animals↗

Influence of infecting serogroup on clinical features of leptospirosis in dogs.

The purpose of this study was to review recent cases of leptospirosis seen at referral centers in New York State and to identify differences in clinical or clinicopathologic aspects of the disease among different suspected infecting serogroups. Medical records at the Cornell University Hospital for Animals and the Animal Medical Center in New York City were reviewed to identify dogs diagnosed with leptospirosis from September 1996 to August 2002. Records of 55 dogs met the inclusion criteria for the study. The suspected infecting serogroups included 21 occurrences of Grippotyphosa, 12 of Pomona, 6 of Autumnalis, 5 of Bratislava, 2 of Hardjo, and 1 of Canicola. Five dogs had equal titers to serogroups Grippotyphosa and Pomona, and 3 had equal titers to 2 other serogroups. Common clinical signs included lethargy, anorexia, and vomiting. Common clinicopathologic findings included anemia, thrombocytopenia, azotemia, hyperphosphatemia, high liver enzyme activity, and hyperbilirubinemia. Forty-three of 55 dogs were discharged from the hospital. Serogroup-specific analysis indicated that dogs with suspected serogroup Pomona infection were more likely to suffer from vomiting (P = .01), thrombocytopenia (P = .009), severe azotemia (P = .04), and hyperphosphatemia (P = .006) than dogs with other serogroups and were less likely to be discharged alive from the hospital (P = .03). This study suggests that only minor clinically relevant differences exist among serogroups. Leptospira serogroup Pomona caused more severe renal disease and was associated with a worse outcome compared with disease caused by other serogroups.

Animals↗

Expression of leptospiral immunoglobulin-like protein by Leptospira interrogans and evaluation of its diagnostic potential in a kinetic ELISA.

The search for novel antigens suitable for improved vaccines and diagnostic reagents against leptospirosis led to the identification of LigA and LigB. LigA and LigB expression were not detectable at the translation level but were detectable at the transcription level in leptospires grown in vitro. Lig genes were present in pathogenic serovars of Leptospira, but not in non-pathogenic Leptospira biflexa. The conserved and variable regions of LigA and LigB (Con, VarA and VarB) were cloned, expressed and purified as GST-fusion proteins. Purified recombinant LigA and LigB were evaluated for their diagnostic potential in a kinetic ELISA (KELA) using sera from vaccinated and microscopic agglutination test (MAT)-positive dogs. Sera from vaccinated dogs showed reactivity to whole-cell antigens of leptospires but did not show reactivity in the KELA assay with recombinant antigens, suggesting a lack of antibodies to Lig proteins in the vaccinated animals. The diagnostic potential of recombinant Lig antigens in the KELA assay was evaluated by using 67 serum samples with MAT > or =1600, which showed reactivity of 76, 41 and 35% to rConA, rVarA and rVarB, respectively. These findings suggest that recombinant antigen to the conserved region of LigA and LigB can differentiate between vaccinated and naturally infected animals.

Amino Acid Sequence↗