Search

Approximately one-third of the reportable USDA Category D and E laboratory animals in the United States are expended on the potency testing of leptospiral vaccines by the codified hamster vaccination-challenge assay. Valid tests require ≥80% of challenge controls to succumb to disease and an LD50 between 10 and 10,000. This work evaluates the risk associated with the removal of LD50 limits; thereby, eliminating back-titration hamsters from in vivo potency assays for Leptospira (L.) serogroups Canicola and Icterohaemorrhagiae. The impact was assessed through 1) retrospective analysis of industry and CVB serial release data from July 2011-April 2015 and 2) evaluation through vaccination-challenge assays. For the initial vaccination-challenge assays (n = 3/serogroup), one group received potent bacterin (PB) and six groups received subpotent bacterins (SB1-SB6). PB and SB1 were challenged with a single dilution of Leptospira between 10 and 10,000 LD50. SB2-SB6 received serial dilutions of more concentrated challenge. Based on the retrospective analysis and in vivo assays, 80% of the challenge controls succumbing to disease reasonably ensured the minimal LD50 was administered. Subpotent vaccines were not at increased risk for being deemed potent when challenged with >10,000 LD50, but potent vaccines were at risk of being deemed subpotent when challenged with >10,000 LD50., (Published by Elsevier Ltd.)

The Leptospira interrogans vaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered in Leptospira interrogans serovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge with L. interrogans serovar Canicola to hamsters. Recombinant LipL32, purified from an Escherichia coli expression system, was assessed for protective immunity in five groups of hamsters (n = 5) following a challenge with the virulent L. interrogans serovar Canicola strain Kito as a challenge strain. However, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts of L. interrogans in the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response against L. interrogans serovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.

Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect) in a microscopic agglutination test (MAT). This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.

Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3 % (69/323), 17.2 % corresponded to household dogs and 26.7 % to stray dogs. Leptospira Canicola (29 %), Leptospira Hardjo (22.58 %), and Leptospira Icterohaemorrhagiae (16.12 %) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78 %), Leptospira icterohaemorrhagiae (13.15 %), and Leptospira Pomona (7.89 %) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor.

The current batch potency test for Leptospira interrogans serovar Canicola vaccines requires the use of a large number of hamsters and has severe effects (i.e., hepatic and renal failure resulting in death); while this vaccine is effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to analyze vaccine proteomes and identify target molecules common to all L. interrogans serovar Canicola vaccines which could be used to design an in vitro potency test. Initial analysis of L. interrogans serovar Canicola vaccines (A to E) from different manufacturers, using the Limulus amebocyte lysate assay and silver-stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not present in all vaccines, preventing it from being a suitable target molecule. The protein contents of vaccines A to E were therefore determined by two-dimensional liquid chromatography mass spectrometry ([2D-LC/MS] 221 ± 31, 9 ± 8, 34 ± 4, 21 ± 5, and 34 ± 17 proteins [mean ± 1 standard deviation] found, respectively). The outer membrane protein LipL32 was established to be common to all and to be present at a significantly higher (P ≤ 0.05) relative spectral abundance in a batch of vaccine which passed the in vivo potency test than in one which had failed. Further analysis using multiple reaction monitoring revealed that the concentration of the N terminus of LipL32 was significantly lower (P ≤ 0.01) in failed batches (n = 2) of vaccine than in passed batches (n = 2); the concentration of the C terminus between the two batches was approximately the same. An in vitro Leptospira vaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Objectives: The aim of the study was to investigate the presence of serum antibodies to different Leptospira serogroups in dogs with a clinical diagnosis of leptospirosis in southern Germany and to compare seroreactivity to different serogroups with history, clinical signs, laboratory findings and survival rate., Methods: In this study, the data of 42 dogs with the diagnosis of leptospirosis were evaluated retrospectively. Dogs were presented to the Small Animal Medicine Teaching Hospital (Medizinische Kleintierklinik) of the Ludwig Maximilians University Munich, Germany, between 1990 to 2003., Results: Reactivity to the serogroup grippotyphosa (13/42) was most frequently present, followed by reactivity to the serogroup saxkoebing (10/42). There was no difference in the clinical picture and the laboratory changes between dogs whose sera were reactive to different serogroups., Clinical Significance: Most of the dogs with leptospirosis in southern Germany had sera reacting to serogroups other than icterohaemorrhagiae and canicola, which are contained in the vaccine. Thus, currently available vaccines in Europe do not protect against the most common Leptospira organisms associated with clinical disease.

By using the monoclonal antibodies methodology we have classified Cuban serovars of leptospires isolated from blood culture of confirmed leptospirosis patients. The serogroups Pomona and Canicola were the most predominant found in our study. Pomona, Mozdok and Canicola serovars were the most prevalent types in these serogroups. Our study has essentially presented a validation of a monoclonal antibody method for the serotyping of Cuban leptospire isolates.

The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.

Leptospira serogroup Ballum is at present the first cause of human leptospirosis in Cuba. Killed whole-cell vaccines were formulated with two clinical isolates of Leptospira interrogans serogroup Ballum using aluminum hydroxide as adjuvant. Agglutinins levels induced by each vaccine in hamsters were estimated by microscopic agglutination test and specific IgG activities were quantified by a whole cell-based enzyme-linked immunosorbent assay. Homologous and cross protective capacity against lethal and sublethal infection were determined in vaccinated animals by challenge with 100 and 10,000 LD50 of five virulent strains belonging to serogroups Ballum, Canicola, Icterohaemorrhagiae and Pomona. Both monovalent serogroup Ballum vaccines were immunogenic and induced complete homologous protection in the animal model. Cross-protection was only significant in one of the two vaccines against challenge with 100 LD50 of serogroup Canicola. The results of this study demonstrate the high immunogenicity and protective capacity in hamsters of whole-cell monovalent vaccines formulated with two vaccine candidate strains belonging to the most prevalent serogroup of Leptospira in Cuba.

Two detergents (triton X-100 and sodium desoxycholate) were evaluated as solubilizing agents of the components of the outer membrane of Leptospira Pomona serovar mozdok. It was obtained a greater yielding of proteins on applying the treatment with triton X-100. The variants formulated starting from the extracts obtained showed a high immunogenicity and capacity of homologous protection against the challenge in hamster.

Two vacine candidate strains from Ballum serogroup were characterized and their immunogenicity and protective power were studied in hamster. The monovalent vaccine formulations obtained showed a high immunogenicity and capacity of homologous protection and, in a lesser degree, of cross protection.

The operational conditions for the production of vaccine vax-SPIRAL were established at the scale of 100 L. It was concluded that it should be carried out with an air flow of 10 L/min (0.1 vvm) and without mechanical agitation.

It was proposed to substitute the present technology of the lavage operation for a tangential microfiltration system at the lavage stages of the culture of Leptospira interrogans canicola canicola. It was proved that this system may be applied, since no cellular affectation was observed and high pollutant elimination levels were obtained.

A quantitative ELISA for the detection of IgG antibodies in response to the Cuban vaccine against the human leptospirosis (vax-SPIRAL) was applied to 930 volunteers'sera, 483 immunized with vax-SPIRAL and 447 with a vaccine against hepatitis B. Samples were taken before beginning the study, 21 days after the second dose and a year later. A high seroprevalence of antibodies to the vaccine serovars was observed before beginning the study. The initial value of the response doubled at 21 days of the second dose in 45 % of the individuals. There were markedly significant differences (p=0,000000) between both groups. A similar response to the three leptospiral serovars was found. After the year the levels of antibodies decreased; however, 23.1% of the individuals were able to duplicate their level of antibodies regarding the initial moment, and the significant difference with the group that received the vaccine against hepatitis B still existed. The application of this Cuban immunogen amomg the main risk groups was recommended.

Duration of immunity in dogs induced with current commercial inactivated leptospirosis vaccines and evaluated against experimental infection, to date, has hardly been documented. The purpose of the present work was to assess the duration of immunity in dogs that is attainable with a commercial inactivated bivalent leptospirosis vaccine. For this purpose, young dogs were vaccinated twice followed by challenge with either Leptospira interrogans serovar canicola or L. interrogans serovar icterohaemorrhagiae 5 weeks, 27 weeks or 56 weeks after the second vaccination. For assessment of the duration of immunity, titres of agglutinating serum antibodies were measured before and after challenge, and the effects of challenge on a variety of parameters were determined including reisolation of challenge organisms from blood, urine and kidney. Both challenge strains induced a generalised infection in control dogs, the canicola strain being most virulent. From the results with different parameters it appeared that the two vaccinations induced a high rate of protection from generalised infection with canicola and icterohaemorrhagiae at 5, 27 and 56 weeks after the second vaccination. In addition, after 56 weeks, still a high level of immunity against renal infection with sv. canicola and, as a consequence, urinary shedding of sv. canicola bacteria, was demonstrated. It was, therefore, concluded that with this vaccine, using this vaccination schedule, a duration of immunity of 1 year can be attained against infection with both serovars.

Since April of 1998 a high number of leptospirosis cases were detected, coming from the area of Reconquista Central Hospital in Santa Fe province. Since January of that year a notable increase in rainfall and river levels was observed causing inundation. As screening test, macroscopic agglutination (MAT) using 10 serotypes of L. interrogans. Among the 122 patients studied 71 were TR positive and 52 were also ELISA positive, leptospirosis diagnosis being confirmed in 40 of them. Five infecting serogroups were identified: Icterohaemorrhagiae (7/40), Ballum (5/40), Sejroe (3/40), Pomona (3/40) and Canicola (2/40). In the remaining cases (20/40), co-agglutinins were found at the same titer against two or more serotypes of leptospires. Infection prevalence was higher in men and productive age (21 to 40 years). The clinical symptoms more frequently observed were headache, fever and myalgias. All cases occurred after the rains and in the period when the area was flooded. Their clinical presentation, time distribution, geographical localization and high frequency of contact with the risk factor inundation could indicate that, independently of search activities, there was an outbreak.

The standard method for the diagnostic of leptospirosis is the microscopic agglutination test (MAT). Nevertheless, it is time-consuming and presents risk of infection for laboratory personal. Several attempts to simplify and reduce the time, skill and expense required for serological testing were made. Rapid slide agglutination tests (RSAT) have been used nowadays as a reliable screening test for the detection of acute and recent infections by many laboratories. The purpose of that study was to evaluate a new antigen preparation for the rapid diagnoses of acute canine leptospirosis and to compare it with MAT results in clinically suspect serum samples. Two hundred and thirteen serum samples from dogs of both sexes and different ages were tested and 141 (66.2%) animals were considered as positive by MAT. The most frequent serovar was icterohaemorrhagiae, followed by canicola. RSAT results were very similar to those observed on MAT, with 139 (65.3%) reactive animals. Correlation between RSAT and MAT was positive (0.82) and significant (p < 0.01). Concordance of results was of 93.4% (199/ 213) of the animals correctly diagnosed by RSAT. Considering MAT as the standard test, sensitivity of RSAT was calculated on 94.3% (133/141) and specificity on 91.7% (66/72).

A controlled double-blind trial was conducted with the participation of 80 adult volunteers of both sexes, who were randomly divided into groups of 40 individuals each one. The case-base study received the vaccine and the control group was administered placebo to know the safety, the behaviour of reactogenicity, and to star the immunogenicity studies of the first Cuban vaccine against human leptospirosis. The vaccine used in the case-base study was an inactivated and trivalent vaccine containing strains of Leptospira canicola, icterohaemorrhagiae and pomona, since they have the highest circulation in the country. The results obtained showed the inocuity of the vaccine as no adverse severe reactions were detected. The general symptomatology observed was low, where as febricula was the most common general symptom. It appeared during the first 3 days of observation and there were no significant differences between the 2 group. Only a mild pain at the site of the injection was reported as a local symptom, which was more frequent in the vaccinated group than in the control group (7.8 against 1.5%, respectively). The seroconversion obtained was of 29% by microagglutination, and of 34.2% by ELISA. The final results allowed to conclude that this vaccine is safe for human adults at the ages under study, and give the possibility to continue other studies in more advanced stages to complete the requirements for obtaining its license.

We determined the seroprevalence of leptospirosis in a suburban canine population for the purpose of analyzing the association between different individual and environmental variables and seropositivity for leptospirosis. The study, which was cross-sectional, was performed in July 1992 in a neighborhood of Greater Buenos Aires with approximately 9,500 inhabitants and a canine population of around 2,000 animals. We studied a random sample of 223 dogs and obtained a blood sample from each. Each animal's epidemiologic history was obtained by interviewing the housewife. Serologic measurements were performed by the microagglutination technique with the use of 10 different serotypes of Leptospira interrogans. Of the 223 dogs that were tested, 57% showed seropositivity; 82% of the positive sera coagglutinated with two or more serotypes. The most frequently detected serotypes were canicola and pyrogenes. Seroprevalence in females was less common than in males (P < 0.05) and in puppies less than 1 year old it was less common than in older animals (P < 0.01). Street behavior in the dog and the presence of stagnant water in front of the owner's dwelling were the most important of the risk factors examined. The associations between seropositivity on the one hand and contact with trash deposits, hunting behavior and the presence of rodents inside the dwelling on the other were not statistically significant. Different control measures are discussed.

Sensitivity and specificity of indirect immunoenzyme method for determining antileptospirosis antibodies have been studied. Supersound antigenic preparations are used as antigens. Determination of genus-specific antibodies and antibodies of more narrow specificity including serogroup specific ones is possible in the elaborated test. A spectrum of revealed serogroup specific antibodies expends in patients with leptospirosis when using polyvalent antigenic preparations.

During the period of 34 years (1957-1990) diseases caused by leptospires of the serogroup canicola were confirmed in 2.7% of the total number of patients. The analysis revealed that in the presence of epidemic outbreaks Leptospira infection caused by bacteria of the serogroup canicola occurred 1.7 times more often than at the time when such outbreaks were absent. Periodic increases of leptospirosis caused by this serogroup were registered in individual towns and regions of the territory in different years. The analysis of 33 medical histories made it possible to state that the clinical manifestations of leptospirosis caused by bacteria of the serogroup canicola were characteristic of Leptospira infections caused not only by this serovar. As a rule, this kind of leptospirosis was registered in rural and urban areas as individual isolated cases.

An enzyme-linked immunosorbent assay (ELISA) was assessed to detect in humans IgM and IgG against the main serogroups/serovars of Leptospira present in Italy. Sonicated antigens from strain Wijnberg, serogroups Icterohaemorrhagiae and Riccio 2, serogroup Australis, were used in ELISA and the results were compared to microagglutination test (MAT). IgM were confirmed to be the predominant class of antibodies; the total sensitivity obtained by Wijnberg/ELISA with the sera of patients infected by different serogroups was 81.5% in relation to MAT, better sensitivity was achieved by the use of Riccio 2/ELISA; no reaction occurred with the control sera. Cross-reactivity was noted with some sera of patients with Lyme disease (IgM) and with few sera of patients with autoimmune disease (IgM and IgG). To improve the sensitivity of the ELISA both antigens Wijnberg and Riccio 2 must be employed in the immunoenzymatic assay, since serogroup Australis must be considered as a new emerging serogroup causing human leptospirosis in our country.

Thirty-seven sera samples from patients with leptospirosis icterohaemorrhagic form were studied with a time interval of 2 to 12 days between the beginning of the symptoms and the collection blood samples. It was isolated leptospira of 5 patients' hemocultures (13.5%) and from 4 of these the etiological agent pertained to the serogroup Icterohaemorrhagiae serovar copenhageni. Thirty-five of them (94.6%), including the four patients whose the etiological agent was isolated, showed reactivity in the enzyme linked immunosorbent (ELISA) IgM test. By this way, it was demonstrated that this test is important for a rapid diagnosis of human leptospirosis, even in the beginning of the disease, when there is still leptospiraemic phase.

An enzyme-linked immunosorbent assay ELISA was evaluated for the detection of IgA antibodies in the human leptospirosis. The assay proved to be sensitive and specific when compared with the ELISA-IgM, in the examined serum samples. The results found suggest that IgA antibodies became positive later in leptospirosis, and will can be an evolutive indicator in the development of the disease.

To study the pulmonary complications in leptospirosis case records of 23 such patients admitted at the Hospital Universitário Antônio Pedro, Universidade Federal Fluminense, Niterói, Brasil, were reviewed. Hemoptysis were seen in 21.7% and sputal blood in 30.4% of patients. Arterial gasometry detected hypoxemia and hypocapnia in most cases. Thoracic radiology showed an alveolar pattern in 60% of the patients, alveolo-interstitial in 20%, interstitial in 6%, and in 14% the lungs were considered to be normal. Necropsy of 13 cases showed edema, congestion and hemorrhage in the lungs in all cases. Hyaline membrane was found in 30% and fibrin thrombi in 46% of these cases, resulting in a diagnosis of adult respiratory distress syndrome and acute disseminated intravascular coagulation (consumption coagulopathy) in leptospirosis.

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28% hexoses), 4% protein, 20% lipid and 2.7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.

A serological survey of goats in St Elizabeth Parish, Jamaica, comprising the breeding herd at the Agricultural Development Corporation (ADC) facility at Hounslow, five commercial herds and 21 family herds was carried out in 1985-1986. The Leptospira microscopic agglutination test indicated that one or more serovars in the Canicola serogroup were the most prevalent. Repeated samplings of goats at ADC, Hounslow demonstrated an increase in portlandvere and canicola seropositive animals following the onset of warmer weather and of icterohaemorrhagiae seropositive animals following the autumn rains and cooler weather. Apart from a cluster of family goats seropositive to pomona there was no geographical separation of reactors to the other serovars tested in the parish.

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.

Sodium dodecyl sulphate extracts of the reference strains Mus 127, Castellón 3 and Arborea of the Ballum agglutinogenic serogroup of Leptospira interrogans (the species of pathogenic leptospira), of strain Patoc 1 of the saprophytic species of L. biflexa, and of strain 3055 of illini serotype, the sole representative of L. illini, were each fractionated by ultracentrifugation in a sucrose density gradient into 10 fractions. The fractions were tested by complement fixation and immunodiffusion against the sera of animals during the process of immunisation and during the course of naturally occurring infections. The fractions could be divided into three main pools of serological activity: pool I (fractions 1, 2 and 3), pool II (fractions 4, 5 and 6), pool III (fractions 7, 8 and 9). Pool I was species/genus specific; fraction 1 tended to be species specific while fraction 2 reacted with antisera to all strains whether pathogenic or saprophytic. Pool II was serogroup specific and reacted only with antisera to members of the same serogroup. Pool III was serotype (serovar) specific and revealed the identity of the infecting strain at an early stage of infection.

Normal serum from the painted turtle (Chrysemys picta), the snapping turtle (Chelydra serpentina), and the frog (Rana pipiens) were found to possess bactericidal activity towards Leptospira. Leptospires from both the parasitic and biflexa complexes were killed by these sera at high dilutions. This pattern differs from that of mammalian serum, as generally only the biflexa complex leptospires are killed by normal mammalian serum. The activity in C. picta serum was characterized as being complement dependent and not mediated by basic proteins. Because comple-inactivated C. picta serum regained leptospiricidal activity after the addition of fresh rabbit serum, antibody is also likely to participate in the killing activity. Further support that C. picta serum contained leptospiral antibodies was found by the detection of serotype-specific agglutinins.

Resistance to clinical and renal leptospirosis in dogs injected with antiserum to Leptospira interrogans serotype canicola and Leptospira interrogans serotype icterohaemorrhagiae was demonstrated. The relationship between resistance induced in serum-injected hamsters was also demonstrated, using criteria of leptospiremia, renal infection, leptospiruria, and clinical signs of disease in dogs and death in hamsters.

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.

The affinity constants of anti-canicola and anti-hebdomadis monoclonal antibodies and the numbers of antigenic determinants per organism of each serovar of leptospira were determined, and their influence on the results of microscopic agglutination test (MAT) and ELISA were examined. In the combinations of a monoclonal antibody and some serovar which showed higher affinity constants at levels of 10(7) to 10(8)/M order, both MAT and ELISA were positive except for one combination, while in the combinations which gave lower affinity constants at levels of 10(6)/M order, MAT was positive but ELISA was negative. The number of antigenic determinants seemed to have no significant influence on the results. The disagreement due to the difference in the threshold affinities of MAT and ELISA should be considered in the use of monoclonal antibodies in the taxonomy of leptospira.

An oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola, recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi, was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.

Obtained was a five-valent vaccine (Leptospira pomona, L. canicola, L. grippotyphosa, L. mitis, and L. icterohaemorrhagiae). It was found that storage at 4degreesC does not impair the immunogenicity of the vaccine in the course of one year. Three groups of rabbits were used to test its immunogenicity--group I, injected once, subcutaneously, with 1 cu. cm of the vaccine; group II, injected subcutaneously, twice, at an interval of 7 days, with 1 cu. cm of the vaccine each time; and group III, injected once, muscularly, with 1 cu. cm. The animals were kept under observation for twelve months. It was established that in the case of intramuscular treatment there were highest antibody titers in the blood serum persisting up to the twelfth month.

Epidemiological and clinical aspects of 50 consecutive patients, 47 adults and three children, hospitalized between 1977 and 1987 for human leptospirosis, were reviewed. 45 (90%) of the patients were from rural regions. 32 (64%) cases occurred in individuals at occupational risk for the infection. 35 (70%) cases were registered in the warm season. The source of infection was known in 34 (68%) cases. Weil's disease was diagnosed in 31 (62%) patients, aseptic meningitis in 12 (24%) and acute unexplained fever in seven (14%). Haemodialysis was required for 11 (35%) patients with Weil's disease. Three (6%) patients died. Cause of death was massive gastrointestinal haemorrhage in two and renal failure in one. Leptospira icterohaemorrhagiae was responsible for 39 (78%) cases, Leptospira canicola for six (12%), Leptospira grippotyphosa for two (4%), and Leptospira australis, Leptospira ballum and Leptospira sejroe, for one case each. A muscle biopsy was performed in six patients and a renal biopsy in three. Focal necrotic muscular changes, with mild mononuclear infiltrate, were found. Pigmented casts in distal convoluted tubules, mild interstitial inflammatory infiltrate and mesangial enlargement of some glomeruli were observed in kidney biopsies. A good knowledge of the protean clinical manifestations of leptospirosis and an accurate laboratory study are required for a correct diagnosis.