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rRNA gene restriction patterns of Leptospira: a molecular typing system.

A total of 67 serovar reference strains and 7 isolates belonging to the genus Leptospira were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns. Fifty patterns were observed. Strains belonging to different genomic species always gave different patterns. However, genomic species were subdivided into several patterns. Forty-three serovars gave a specific pattern. Some serovars could not be separated by rRNA gene restriction patterns: strains of serovars icterohaemorrhagiae, copenhageni, lai, pyrogenes and jalna gave pattern 1; serovars birkini, mankarso and wolffi gave pattern 4; serovars canicola, gem, hebdomadis, pomona and hardjo (strain hardjoprajitno) gave pattern 12; serovars valbuzzi and zanoni gave pattern 14; serovars jonsis, malaya and sumneri gave pattern 16; serovars arborea, ballum, castellonis and kenya gave pattern 35; and serovars borincana and shermani gave pattern 43. These data provide the bases for a molecular typing system for the genus Leptospira.

Bacterial Typing Techniques↗

The occurrence of leptospirosis in Iran.

The author examined 2,448 human and 541 domestic animal sera from Iran serologically for leptospira antibodies. Human sera revealed a 3.3% positivity rate in titer levels of 1:100 and higher, namely with the serovare of the serogroups Australis 2.3%, Sejroe 0.8%, Grippotyphosa 0.2%, Icterohaemorrhagiae 0.1%, Canicola 0.1%, Autumnalis 0.4% and Javanica 0.04%; domestic animal sera revealed a 6.8% in titers of 1:800 and higher, namely with Icterohaemorrhagiae 3.8%, Grippotyphosa 1.4%, Sejroe 1.2% and Bataviae 0.4%. The positivity rate for men was 3.1% for women 3.6%. In Teheran the seropositivity was higher--3.6% as in Rasth, the capital of the Gilan province--1.9%.

Age Factors↗

[Survey of anti-Leptospira agglutinins in workers from the city of Londrina-Paraná, Brazil].

Serum samples were obtained from 249 individuals in Londrina-Paraná, Brazil. The sera were analysed for leptospiral agglutinins by microscopic agglutination tests and 22.1% were positive. Of these positives per group were 24.6% for butchers, 25.9% for farm workers, 17.2% for children and 22.2% for servants. The agglutinin titers ranged from 1:200 to 1:3200. The highest titers were obtained for the serovars sejroe (butchers), grippotyphosa (farm workers), javanica, canicola, panama, wolffi and pyrogenes (children) and butembo (servants).

Abattoirs↗

Effect of heat or formalin treatment of leptospires on antibody response detected by immunoblotting.

Leptospira interrogans serovar icterohaemorrhagiae RGA (RGA), live or heated at 56 degrees C for 15 min or treated with Formalin, was injected into rabbits to prepare hyperimmune serum. The pathogens L. interrogans serovars icterohaemorrhagiae RGA, icterohaemorrhagiae 1, canicola Moulton, grippotyphosa Andaman, hardjo Hardjoprajitno, and pomona Pomona and the nonpathogen Leptospina biflexa serovar patoc Patoc 1 were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and after electrophoresis they were then transferred to nitrocellulose paper. Antiserum against RGA (live, heat killed, or Formalin killed) was used on one of each of the three blots. Formalin appeared to completely eliminate antibody response to antigens with the molecular weight of 14,000 to 20,000 (14K to 20K) but did expose an antigen at approximately 23K in the pathogens only. This same band had only slight reactivity when antiserum against heat-killed RGA was used. Heating also eliminated cross-reactivity in the 19K to 30K range and partially degraded bands in the 14K to 20K region so that one broad band rather than several discrete bands appeared. The three antiserum specimens cross-reacted with all of the serovars tested, but fewer antigens of grippotyphosa and hardjo reacted with the antisera. Against patoc, reactivity was limited primarily to the flagellar region. The most cross-reactivity was with the antiserum prepared by using live leptospires.

Animals↗

Leptospiral abortion and leptospiruria in horses from the same farm.

Leptospirosis was documented as the cause of abortion in a 5-year-old mare. Leptospires were detected in tissue specimens from fetal kidneys and from placenta by histologic evaluation of silver-stained sections. Antibodies against Leptospira interrogans serovar pomona were detected in fetal serum at a titer of 1,600 by use of a microscopic agglutination test. The mare had serum titers of 6,400; 0; 400; 800; 3,200; and 6,400 to L interrogans serovars bratislava, canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona, respectively. A serologic survey identified titers of at least 6,400 against serovars bratislava and pomona in 5 other horses on the farm. Titers of at least 100 against serovar bratislava were detected in 53% of the horses on the farm. Leptospires were detected by direct fluorescent-antibody testing in urine samples from the mare that aborted and from 2 of the other 5 horses.

Abortion, Veterinary↗

Serological studies and isolations of serotype hardjo and Leptospira biflexa strains from horses of Argentina.

Three pathogenic leptosipras and 12 saprophytic Leptospira biflexa strains were isolated from 72 apparently normal horse kidneys collected at an abattoir in Argentina. Cross-agglutination reaction patterns of the pathogens showed that they were antigenically homologous with members of the Hebdomadis group. When one of the strains was compared to Hebdomadis serotypes in reciprocal agglutination-absorption tests, it was found to be serologically homologous to serotype hardjo. This is the first known report of an isolation of this serotype from horses. Serological tests were also carried out on randomly collected abattoir sera from 245 horses to determine the prevalence of equine leptospirosis. Significant antibody titers (1:100 or greater) were found in 74.6% of the sera. Predominant reactions occurred with the antigens pomona, hebdomadis group, pyrogenes, tarassovi, and canicola. Agglutination tests performed with antigen prepared with one of the saprophytic biflexa isolates showed seropositive reactions in 99.1% of the equine sera, with agglutination titers ranging from 1:100 to 1:3,200. Absorption of selected horse sera with the saprophytic strain removed the agglutinins to Leptospira interrogans serotypes. This suggests the possibility that L. biflexa strains may act as an antigenic stimulus and account for some of the persistent multiple cross-reaction patterns of equine sera with pathogenic serotypes.

Abattoirs↗

Clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs.

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay on urine samples for diagnosis of leptospirosis in dogs. DESIGN: Prospective case study. ANIMALS: 132 dogs with clinical signs suggestive of leptospirosis and 13 healthy dogs. PROCEDURE: PCR testing was performed on urine samples to detect leptospiral DNA; results were compared with results of conventional criteria for the diagnosis of leptospirosis. RESULTS: Leptospirosis was diagnosed in 8 dogs via established criteria; all these dogs had positive results of PCR assay, including 1 dog with positive results before seroconversion developed. A positive PCR assay result was also obtained in 16 dogs that did not have a confirmed diagnosis of leptospirosis. In the 8 dogs that had a confirmed diagnosis of leptospirosis, serovars pomona (n = 3 dogs), grippotyphosa (2), canicola (2), and bratislava (1) were identified serologically. The remaining 121 dogs all had a diagnosis other than leptospirosis or were healthy. For PCR testing on urine, sensitivity was 100%, specificity was 88.3%, positive predictive value was 33%, and negative predictive value was 100%. CONCLUSIONS AND CLINICAL RELEVANCE: Positive PCR test results prior to seroconversion may have value in establishing an early diagnosis. Positive results in dogs that had signs consistent with leptospirosis despite failing to meet established criteria for leptospirosis raise questions regarding the sensitivity of serologic testing in diagnosis of leptospirosis. Serovars pomona, grippotyphosa, and canicola were most common.

Animals↗

Treatment and outcome of dogs with leptospirosis: 36 cases (1990-1998).

OBJECTIVE: To characterize serologic and clinical features and outcome of dogs with leptospirosis that were treated conservatively (i.e., medical management alone) or with hemodialysis. DESIGN: Retrospective study. ANIMALS: 36 dogs with leptospirosis. PROCEDURE: History; results of physical examinations, ultrasonography, and serologic, hematologic, and serum biochemical analyses; time to resolution of azotemia; and outcome were obtained from medical records. Dogs were treated conservatively (n = 22) or with hemodialysis (14). RESULTS: Between 1990 and 1998, amount of rainfall was positively correlated with number of cases of leptospirosis identified per year. Serum antibodies against 6 Leptospira serovars were measured, and titers were highest to Leptospira pomona in 16 (44%) dogs, L bratislava in 9 (25%) dogs, and L hardjo in 1 (3%) dog. Eight (22%) dogs had equally high titers to L pomona and L bratislava, 1 (3%) had equally high titers to L grippotyphosa and L canicola, and 1 (3%) had high titers to L grippotyphosa, L pomona, L canicola, and L bratislava. During initial evaluation, all dogs were azotemic. Thirty (83%) dogs survived, including 12 of 14 (86%) dogs treated with hemodialysis and 18 of 22 (82%) treated conservatively. Serum creatinine concentration was similar in both groups after resolution of clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with L pomona and L bratislava was recognized as a cause of leptospirosis in dogs, and resulted in development of acute renal failure with various degrees of azotemia. Prognosis for dogs with mild to moderate azotemia was good with conservative treatment, whereas treatment with hemodialysis appeared to improve prognosis for dogs with severe azotemia.

Animals↗

Leptospirosis in patients from Grenada, West Indies.

Evidence of leptospirosis in humans from Grenada, W.I., is reported here for the first time. Of 139 hospital patients with pyrexia of undetermined origin, 94 were jaundiced, and 16 (17%) of these were positive for hepatitis B antigen (HBsAg) and not examined further. 20 of the remaining 123 (16%) febrile patients were positive for leptospiral antibodies; 11 of them were current or recent cases. 25 of 182 (14%) afebrile out-patients showed evidence of previous exposure to leptospiral organisms. The most frequently encountered serogroups in the 45 seropositive in- and out-patients were Icterohaemorrhagiae (38%), Panama (24%) and Canicola (11%).

Adolescent↗

Epidemiologic features of equine Leptospira interrogans of human significance.

Leptospirosis is a zoonotic bacterial disease caused by Leptospira interrogans. There is a serologic evidence that horses are exposed to L. interrogans and, as a shedder of these organisms, can be a threat to humans. We examined risk factors associated with the risk of testing seropositive to three L. interrogans serovars (L. icterohaemorrhagiae, L. grippotyphosa, and L. canicola) in the horses of New York State, in order to understand the epidemiology of the disease and suggest strategies to control and prevent equine leptospirosis. To carry out this study, blood samples were collected from a random sample of 2551 horses and tested for the presence of antibodies to the above serovars using the microscopic agglutination test. Samples with a titer $100 were considered positive. Clinical and demographic data were collected on each horse, the farms' management practices and ecology. Logistic regression analysis was used to develop a multivariate indexing system and to identify factors significantly associated with the risk of leptospirosis. Four indices were developed based on the possible sources of exposure: rodent exposure index; wildlife exposure index; soil and water index; and management index. The soil and water index was significantly associated with the risk of exposure to all three serovars. Management was positively associated with L. icterohaemorrhagiae and L. canicola. Density of horses turned out together was positively associated with the risk of exposure to L. grippotyphosa. We concluded that indirect exposure of horses to L. interrogans through contaminated soil and water appears to be significantly associated with the risk of exposure to all three serovars. Management appears to play an important role in the exposure to L. interrogans. Modification of management practices might reduce the horses' risk of exposure and hopefully minimize the human hazards.

Animals↗

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool.

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in São Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.

Agglutination Tests↗

Epidemiology of leptospirosis: observations on serological data obtained by a "diagnostic laboratory for leptospirosis" from 1995 to 2001.

Serological data on leptospira infection were reported and discussed. From 1995 to 2001, the blood serum samples of 9885 domestic and wild animals and humans, living in Northern and Central Italy, were examined by the macroagglutination test (MAT) employing bratislava, ballum, canicola, grippotyphosa, icterohaemorrhagiae, pomona, hardjo and tarassovi serovars as antigens. Considering sera with > or = 1:400 antibody titers as positive, 674 (6.81%) animals scored positive. Sheep, horses, pigs and dogs gave the highest number of positive responses, particularly against the serovar bratislava and, for dogs, against icterohaemorrhagiae. The percentages of seropositivity observed in the most important animal species were: 12.13% in ovine (132 positive among 1088 tested animals), 11.40% in horses (107 positive animals among 938), 9.46% in swine (123 positive animals among 1299), 6.36% in dogs (278 positive animals among 4369), 2.39% in wild boars (11 positive animals among 459), 1.39% in deer (2 positive animals among 143), 0.48% in cattle (3 positive animals among 626). Among 250 human sera examined, 14 (5.60%) scored positive.

Animals↗

[Immunodiagnosis of human leptospirosis by ELISA-IgM, employing different antigenic preparations from prevalent serovars of Leptospira interrogans].

A comparative study among different serovars of Leptospira interrogans was performed in order to prepare antigens to detect IgM antibodies by ELISA in early and late phase of human leptospirosis. Ten serovars were chosen among the most prevalent detected by microscopic seroagglutination (SAM) in São Paulo city. Using ELISA-IgM five of them showed better results (canicola, hebdomadis, icterohaemorrhagiae, cynopteri and brasiliensis). These ones were also studied in a pool. The non-treated antigens showed higher reactivity than the Triton X-100 (4%/50 degrees C/4h). ELISA-IgM using individually or pool of non-treated antigens proved to be reliable with high sensitivity and should be used for an earlier diagnosis of leptospirosis, as a trial test. Faster diagnostic elucidation can be useful to detect epidemic situations, so, allowing epidemiological surveillance interventions.

Antigens, Bacterial↗

Leptospiral agglutinins in dogs in Sydney.

From June 1971 to June 1972, sera from 600 dogs in Sydney were tested for leptospiral agglutinins by a rapid slide agglutination method. The end-point titre was taken at 50 percent agglutination of the live organisms. Forty-one samples (6.8 percent) had a significant leptospiral titres (100 or greater) and 5 of these reacted to 2 serotypes. Thirty serums (5 percent) contained agglutinins against L. copenhageni, and 6 (1 percent) against L. pomona, while a few samples reacted against hardjo, tarassovi, australis, grippotyphosa or pyrogenes serotypes. No significant titres were found to L. canicola, L. hebdomadis, L. autumnalis or L. bataviae.

Agglutinins↗

Serologic evidence of equine leptospirosis in the northeast United States.

Serologic testing for leptospiral antibody was conducted with the macroscopic agglutination test on 1,346 equine serum samples. These were collected from clinically normal horses in 123 purebred herds in the Northeast. Sixty-eight samples (5%) from the population tested reacted at significant levels (1:40 or higher) to one or more of the 5 serotype antigens used. These reactors were from 38 (31%) of the herds tested. Reactions to serotype pomona predominated in 25 (72%) of these 38 herds. Smaller numbers of herds had reactors to canicola, icterohemorrhagiae and grippotyphosa. No significant reactions to serotype hardjo were detected.

Animals↗

Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays.

Genetic variability among Leptospira interrogans (sensu stricto) serovars was assessed by Southern blot hybridization and PCR analyses. The experiments used probes directed to sequences in a recently described insertion element, IS1500. Hybridization analysis showed that IS1500 was present on polymorphic fragments and that differences in these patterns could be used to identify serovars. Hybridization analysis was also useful in discriminating between serovar pomona type kennewicki isolates, making possible the identification of 15 previously unrecognized genetic groups. A PCR assay was developed in which the primers are positioned near the terminal inverted repeats of the element and directed outward. This assay yielded characteristic amplification patterns from isolates, allowing them to be identified. We applied these assays to several new animal isolates of L. interrogans from Nicaragua, which recently had an outbreak of human leptospirosis. Three groups of isolates were identified: one strain of serovar pomona type kennewicki and two genetically distinct groups of isolates which may be genetic intermediates between serovars canicola and portlandvere. The IS-based typing assays described should be useful for epidemiological analysis of leptospirosis.

Animals↗

[Diagnostic serology of swine leptospirosis in Mexico 1995-2000].

Results obtained from sample testing of 1970 swines from a number of Mexican farms were analyzed. Such samples had been received in the Leptospira Lab of Universidad Autonoma Metropolitana de Xochimilco from 1995 to 2000. Sera with titers equal to or higher than 1:1000 were considered positive; 39,8% of the animals were seropositive (784) and the most frequent serovarieties were bratislava, 22.5%; icterohaemorrhagiae strain Palo Alto, 14,5%; portland vere strain Sinaloa ACR, 13,8%; icterohaemorrhagiae, 11,1%; grippotyphosa, 8,9%; hardjo strain H89,7.2%; tarassovi,7.1%; panama, 5.8%, pomona and hardjo, 5.1%; wolffi, 3%; shermani, 2.4%; pyrogenes, 1.2%; canicola, 0.8%; hebdomadis, 0,5%. The bratislava serovariety has been reported as the cause of reproductive failure in several countries and it holds the first place in serological studies. Therefore, the present paper provides information for stating that this is one of the most significant serovarieties in Mexico.

Animals↗