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From 1970 through 1973, blood samples of 7113 attended dogs and 1615 stray dogs in 50 cities of Japan were examined for leptospiral antibodies by RMAT on filter paper specimens. Results were as follows: 1. Prevalences of antibodies against L. icterohaemorrhagiae and L. canicola in attended dogs in 50 cities ranged from 22.5% to 0% with an average of 9.8%. Those of stray dogs in 8 cities ranged from 52.5% to 0% with an average of 21.7%. 2. 64 and 37 of 7113 attended dog sera reached positively only against either L. icterohaemorrhagiae and L. canicola, respectively. 595 of 7113 attended dog sera reacted positively against both antigens. 3. In 820 attended dog sera tested against 8 leptospiral serotypes, 57 had antibodies against L. icterohaemorrhagiae, 52 against L. canicola, 2 against L. australis A, 10 against L. pyrogenes and 2 against L. grippotyphosa. 4. The prevalence of antibodies was twice as much in male dogs as in females. The prevalence in a healthy group was only half that of a sick group. Attended dogs below 4 years old had a higher antibody prevalence than any other age. The prevalence of antibodies in summer was lower than for any other season.

The occurrence of leptospirosis in swine of southeastern Alabama was determined. A total of 627 sera were tested, using the microscopic agglutination method, with live antigens of 12 serovars. Of the sera tested, 121 (19.3%) had a titer of 1:100 or greater to the serovars employed. The percentage distribution of sera with titers of greater than or equal to 1:100 among serovars most commonly reported was as follows: Leptospira interrogans serovars pomona, 3.8%; icterohaemorrhagiae, 3.3%; canicola, 1.6%; hardjo, 0.7%; and grippotyphosa, 0.16%. Of the less commonly recognized leptospiral serovars, the percentages reacting were as follows: ballum, 4.9%; autumnalis, 3.2%; pyrogenes, 1.1%; and bataviae, 0.4%. None of the sera reacted with antigen of serovars australis, tarassovi, or wolffi.

The comparative evaluation of the interaction of L. icterohaemorrhagiae strain P, L. canicola strain CL and L. hebdomadis strain 650 with golden hamster liver and kidney cells is presented. Three variants of the course of Leptospira infection have been distinguished: (1) the hepato-renal (icteric) variant, caused by the adhesion of leptospires to liver cells with the colonization of their surface and the disaggregation of liver-cell complexes and by the accumulation of leptospires in the kidney interstice; as a consequence, parenchymatous hepatitis and nephroso-nephritis develop, which lead to the death of animals; (2) the renal (anicteric) variant, characterized by the absence of the infective agent and lesions in the liver, by adhesion of leptospires to and their colonization of the nephrothelium of the proximal convoluted tubules of the kidneys; in this case some of the animals die because of renal insufficiency and shock, while in the surviving animals prolonged carrier state develops; (3) the intermediate variant, characterized by the initial process of leptospiral adhesion and colonization in the liver and its subsequent progress in the kidneys.

In order to assess the clinical, laboratorial and epidemiological aspects of feline leptospirosis, ten female and male adult cats were experimentally inoculated with pathogenic and autochthonous field isolate of Leptospira interrogans. Five of them were inoculated subcutaneously with serovar icterohaemorrhagiae (R-192) and the others five with serovar canicola. No clinical and laboratorial alterations were found in these animals. Antileptospiral agglutinins were detected in 90% of the infected cats, shortly after the 1st week after inoculation. The leptospiral agglutinins were detected for 8 to 12 weeks and the elimination of leptospires through urine was observed only in animals infected with serovar canicola, beginning 2 to 4 weeks after inoculation and lasting for 2 to 8 weeks. Isolations of leptospires from blood and kidneys were unsuccessful.

The epidemiology of canine leptospirosis has been examined in a limited study in the Netherlands from 1969 through 1982. Leptospira interrogans serotype icterohaemorrhagiae and canicola were found to be the only serotypes causing clinical leptospirosis. However, positive agglutination titres to the serotypes grippotyphosa, bratislava, poi and ballum have also been detected. The incidence of infection caused by serotype icterohaemorrhagiae was highest during summer and autumn. Infections with serotype canicola were more evenly distributed over the year with only a slight increase during summer and autumn. Infections caused by serotype canicola were significantly more prevalent in male dogs, whereas infections with serotype icterohaemorrhagiae were found both in males and females in almost equal numbers. The incidence of infections with serotype icterohaemorrhagiae was highest in immature dogs. Infections with serotype canicola were detected in young as well as older animals. In contrast to the situation in the past, infections with serotype icterohaemorrhagiae are now more common than those caused by serotype canicola, which have become rare. The highest incidence of infection was found in the western part of the country. The majority of infections was demonstrated in guard-dogs and sporting-dogs. Dogs proved to be of minor importance as a source of human infection in the Netherlands.

The humoral immune response of dogs vaccinated with leptospiral pentavalent outer envelope and whole culture vaccines was monitored with the microscopic agglutination (MA) test and leptospiricidal activity (LA) test for a 2-year period. The leptospiral serovars in the vaccines was canicola, icterohaemorrhagiae, grippotyphosa, pomona, and hardjo. The LA test was markedly more sensitive than the MA test for detecting anti-Leptospira antibodies and was least noticeable with anti-pomona antibodies. Both Igm and IgG were produced in similar amounts in the vaccinated dogs, and both classes of immunoglobulins were reactive in the MA and LA tests. The IgM class of antibodies was slightly more reactive in the MA test, whereas IgG antibodies were somewhat more reactive in the LA test. A direct correlation between protective antibodies, as determined by the hamster passive-protection test, and antibodies reactive in the MA and LA tests was observed.

During 2 years (fiscal years 1973 and 1974), microscopic agglutination tests were performed on 12,565 serums from cattle, swine, horses, deer, sheep, and goats for the detection of leptospiral antibodies. The most frequent presumptive infecting serogroups were Hebdomadis, Pomona, Autumnalis, Ballum, Australis, and Canicola.

Sera from 153 wild animals of 18 species were tested for antibodies against 12 serovars of Leptospira by the microscopic agglutination (MA) test. Seventy-five percent of the animals tested were seropositive against one or more of the 12 serovars used. The most commonly found serovars were pomona, autumnalis, pyrogenes, icterohaemorrhagiae, australis, and canicola. Of 62 carnivores representing 7 species, 55 (89%) were seropositive, as were 46 (60%) of 77 rodents from 9 species. Leptospira of the serovar copenhageni serogroup icterohaemorrhagiae were recovered from kidney tissues of a Norway rat (Rattus norvegicus). Of 443 wildlife sera tested by the indirect hemagglutination (IHA) test using cells sensitized with L. illini antigen, 47 (11%), mainly carnivores and deer, gave a heterophile reaction. Of the remaining 396 sera, 164 (41%) were seropositive for leptospirosis by the IHA test. To compare the IHA test with the MA test, 143 serum samples were tested by both methods. There was 84% concordance between the two tests.

FRom 1970 through 1973, the author used RMAT to examine the canine leptospiral antibodies against 10 serotypes in 12,374 attended dogs, 1,855 stray dogs and 480 house dogs in 24 countries. Among the 10 serotypes, L. icterohaemorrhagiae and L. canicola were the most prevalent. Positive rates were in the following order: Peru 26.3%, Chile 18.4%, Turkey 17.4%, Austria 16.0%, Korea 15.0%, Cina (Taiwan) 11.4%, Japan 9.8%, Brazil 7.7%, United States 5.5%, United Kingdom 4.5%, Italy 3.5%, Germany 2.0%, and Canada 0.4%. Negative results were obtained in Australia, Dominica, Ecuador, Ethiopia, Fiji, Indonesia, Iran, Malta, Philippines, Sweden and Thailand. Positive rates on different continents were: South America 11.1%, Asia 10.1%, Europe 3.4%, North America 2.4%, Oceania and Africa (Ethiopia) 0%. Negative results were obtained against either L. icterohaemorrhagiae or L. canicola in samples from most tropical countries. Lower positive rates were seen during summer in Korea and Japan. Positive reactors were detected against other serotypes, viz., L. grippotyphosa, L. australis A, L. pyrogenes, L. bataviae, L. autumnalis, L. hebdomadis and L. javanica. Sex, physical condition and vaccination status as factors in the presence of antibodies were reviewed.

Resistance to clinical and renal leptospirosis in dogs vaccinated with a bacterin containing Leptospira interrogans serotype canicola and Leptospira interrogans serotype icterohaemorrhagiae was demonstrated. The relationship between resistance induced in vaccinated dogs and that induced in vaccinated hamsters was also demonstrated, using criteria of leptospiremia, renal infection, leptospiruria, and clinical signs of disease in dogs and death in hamsters.

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.

The comparative study of 16 Leptospira cultures, serogroup Canicola, isolated from humans and animals in different years in the North-West of the RSFSR and 2 reference strains of the Canicola serovar, Hond Utrecht IV and Kashirsky, was carried out in the agglutinin cross-adsorption tests. The absence of the antigenic homogeneity of the cultures under test was established: 9 of them proved to be identical to strain Kashirsky and 7, to strain Hond Utrecht IV.

A total of 14 cases of leptospirosis in children aged from 5 to 15 years, hospitalized in the Paris region, are reported. The polymorphe character of the disease is pointed out. The necessity for respecting a rigorous chronological order when requesting complementary examinations, which could confirm the diagnosis, is stressed. Domestic animals, team-mates, and while swimming at week-ends or during holidays, are potential sources or means of infection in the young. This justifies searching for leptospirosis in cases of unexplained fever, a pseudo-influenza syndrome, a meningitis which is mainly of the lymphocytic type, or more rarely, in cases of jaundice. Leptospirosis should be a disease well-known by the pediatrician.

The humoral immune response of cattle vaccinated with leptospiral pentavalent outer envelope and whole culture vaccines was monitored with the microscopic agglutination (MA) test and the leptospiricidal activity (LA) test for a 2.5-year period. The serovars present in the vaccines were canicola, icterohaemorrhagiae, grippotyphosa, pomona, and hardjo. The LA test had greater sensitivity than the MA test for detecting antibodies to the hardjo, canicola, and icterohaemorrhagiae components of the pentavalent vaccine and was approximately equal in sensitivity to the MA test for detecting antibodies to the grippotyphosa and pomona components of the leptospiral vaccine. Both IgM and IgG were produced in similar amounts in the immunized cattle, and both classes were reactive in the MA and the LA tests. The IgM class of antibodies was more reactive in the MA test, whereas the IgG class of antibodies was more reactive in the LA test. Using the MA test as the criterion of evaluating, the immunogenic potency of the individual vaccine components was found to be canicola more than icterohaemorrhagiae more than grippotyphosa more than pomona more than hardjo. A direct correlation between protective antibodies, as determined by the hamster passive protection test, and antibodies reactive in the MA and the LA tests was observed.

Between February 1977 and September 1982, sera from febrile patients from all areas of Trinidad were examined for leptospiral agglutinins; 158 of 1714 patients (9%) were confirmed as current cases of leptospirosis (annual average 26 or 2.6 per 100,000 population). Of the remaining 1556 patients only 711 provided paired sera; 167 of these (23%) showed evidence of previous leptospiral infection. Of the 845 single samples, 125 were positive. The true incidence of the disease is probably much higher than reported here. Males comprised 74% of the current cases; their highest rate of infection was in the 30-39 year age group, the incidence in late teenage approaching this rate. For females, incidence increased with age up to late middle-age. The presumptive infecting serogroups most commonly recorded among the 158 current cases were Icterohaemorrhagiae (37%), Canicola (16%) and Autumnalis (11%). 13 of the cases died (case fatality rate, 8.2%).

Serovars icterohaemorrhagiae, canicola, and arboreae of pathogenic Leptospira interrogans and serovar patoc of saprophytic L. biflexa were examined by crossed immunoelectrophoresis. A close antigenic relationship was found between the interrogans serovars, particularly between icterohaemorrhagiae and canicola. To a much lesser extent cross-reactions were found between interrogans serovars and patoc. Comparison of three different antigenic preparations of icterohaemorrhagiae by tandem crossed immunoelectrophoresis and by absorption experiments with the patoc reference system showed the presence of several common broadly reactive antigens. Antigen 1 of the reference system, a genus-specific heat-stable protein antigen, was found in all antigenic preparations. It is a diagnostically important antigen.

Serological surveys of leptospiral antibodies in cattle were carried out in Macon and the surrounding counties of East Central Alabama. A total of 286 bovine serum samples were screened for the presence of antibodies against live antigens from twelve pathogenic leptospiral serotypes using a microscopic agglutination test. The most frequently encountered serotypes were Leptospira hardjo (47%), Leptospira wolffi (34%), Leptospira canicola (12%), Leptospira pomona (10%) and Leptospira ballum (10%). Leptospira autumnalis, Leptospira grippotyphosa, Leptospira icterohemorrhagiae, Leptospira pyrogenes and Leptospira tarassovi were observed in less than 5% of the samples.

The serovar-specific main antigen (TM antigen) of Leptospira interrogans serovar canicola, which as lipopolysaccharide properties, was treated with 0.1 N sodium hydroxide. This treatment degraded the antigen into two major antigenic components, one of high and one of low molecular weight. The component with the lower molecular weight (approximately 4,000 daltons) consisted mainly of carbohydrates, having lost almost all of the fatty acid and protein components of the original antigen. Although the substance lacked immunoprecipitable activity, it continued to show serovar-specific inhibitory potency in a radioimmunoassay system as well as in a microscopic immunoagglutination reaction of the organisms. The antigenic activity of the compound was also reduced by periodate oxidation as was that of the TM antigen. A component with the same chemical and physicochemical properties was also produced by alkaline treatment from a different serotype TM antigen (serovar kremastos Kyoto), but it showed no antigenic activity.

Investigation of human leptospirosis caused by the serotype canicola revealed a significant shift in the chain of infection of this disease. The adaptation of an old and well-established serotype to a new and abundant host, the Norway rat, provides a new means for spreading and transferring canicola fever to man and farm animals. The changes in the epidemiology of leptospirosis described should serve as a warning to epidemiologists responsible for suggesting proper prophylactic measures.

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect specific anti-leptospiral IgM and IgG in sera of dogs experimentally infected with Leptospira interrogans serotype canicola are reported. In all dogs specific anti-leptospiral IgM was detected from the second half of the first week after infection, the maximum being attained during the second week. Subsequently the IgM titre gradually decreased. Specific anti-leptospiral IgG was detected later and increased gradually to reach almost the same level as the IgM titre after two to three months. During the initial stage of the infection, when the microscopic agglutination titre was still negative or very low, a high IgM titre was accompanied by a negative or very low IgG titre in every case. After the initial stage a substantial IgG titre was also detectable. It is suggested that the test is suitable for serodiagnostic purposes, particularly for the diagnosis of a current infection in an individual.

A total of 597 pigs were examined for serological and bacteriological evidence of leptospiral infection. The sample comprised 189 porkers, 205 baconers and 203 sows. Sera were tested against 17 antigens from 16 serogroups and the overall prevalence of titres was 15.7 per cent. The lowest prevalence of titres was found in porkers (10.0 per cent). The highest prevalence of titres to an individual antigen was recorded with bratislava, a member of the Australis serogroup, with 12.2 per cent of pigs positive. Copenhageni titres were present in 1.8 per cent of the sera and sporadic titres were recorded to various other antigens. Leptospires were isolated from five pigs. Four isolates were identified as belonging to the Icterohaemorrhagiae serogroup and one to the Hebdomadis serogroup. The serological to bacteriological ratio for the Icterohaemorrhagiae serogroup was 11:4. The virtual absence of titres to pomona and tarassovi, the two serovars most commonly maintained by porcine populations in other countries, confirms the results of earlier serological surveys.

Dogs vaccinated with a Leptospira interrogans vaccine containing serogroups Canicola and Icterohaemorrhagiae and prepared from cultures grown in a protein-free medium resisted challenge with heterologous representatives of these serogroups. In contrast, control dogs were pyrexic and leptospires were isolated from the blood for nine days following Canicola challenge and six days after Icterohaemorrhagiae challenge. Leptospires were isolated from the urine of controls throughout the post challenge period and from kidneys at post mortem examination of six out of six and four out of six Canicola and Icterohaemorrhagiae challenged controls, respectively. There have been no reports of hypersensitivity reactions to this vaccine since its introduction in 1980.

The study of DNA in parasitic leptospirae of different serovariants by the method of molecular hybridization has revealed two groups differing in their homology level. The homology of nucleic acids has been found to correlate with serological properties. The serovariants ensuring cross immunity are characterized by the high level of homology of their DNA.

A fluid adjuvanted vaccine consisting of inactivated hepatitis virus (iH) and leptospirae antigens (L) was developed. The vaccine (Kavak iHL; Duphar) was tested in several vaccination programmes both alone and in combination with freeze dried measles (M) or distemper (D) vaccines. The results demonstrate that this new vaccine is also effective in pups with maternally derived antibodies, although a second vaccination at 14 weeks of age is recommended to boost the first vaccination. For the booster vaccination either the iHL-vaccine or the liver attenuated hepatitis vaccine (H) can be used.

A leptospiral genus-specific protein antigen (GP-Ag) was partially purified from Leptospira interrogans serovar kremastos strain Kyoto and canicola strain Hond Utrecht IV, by treating the organisms with Triton X-100 followed by a purification procedure including fractionation with DEAE-cellulose column chromatography and ethanol precipitation. Genus-specificity of the antigens from kremastos Kyoto and canicola was shown by the immunodiffusion (ID) test. Complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA) using GP-Ag from kremastos Kyoto showed the genus specificity of the GP-Ag. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the antigens from kremastos Kyoto and canicola showed a major band with Coomassie blue stain. The band of GP-Ag from kremastos Kyoto was also shown by immunoprecipitation using the monoclonal antibody (GP-7) to GP-Ag from kremastos Kyoto. The molecular weight of the genus-specific antigen was estimated to be approximately 62 000. The genus specificity of the antigen was reduced by heating at 100 degrees C or treatment with proteolytic enzymes, but not reduced by treatment with sodium metaperiodate.

Serological data on the prevalence of human leptospiroses in certain regions of Brazil are presented. Out of 467 diseased and clinically healthy persons, 40 were positive in the Amazonia. The most frequent serotypes were grippotyphosa (27.5%), panama (25%), icterohaemorrhagiae (10%) and woffi (10%). In 1966 and 1970, 279 cases were identified in Recife (northeastern Brazil) during outbreaks subsequent to floods. Among these 92.5% belonged to the icterohaemorrhagiae serotype. From 1947 to 1972, in São Paulo City (southeastern Brazil), of 18,233 patients with clinical signs of leptospirosis, 2,237 were positive with 86.5% belonging to icterohaemorrhagiae. In all Brazil, 32 strains of leptospires were isolated, 27 of which belonged to the icterohaemorrhagiae serotype and one strain for each wolffi, canicola, grippotyphosa, andamana and alexi serotypes.

The immunogenicity of 2 leptospiral cell structural components, the outer envelope (OE) and protoplasmic cylinder (PC), as well as the leptospire whole cell (WC), was compared in hamsters and dogs. The 50% protective dose for hamsters against death (PD50D) and kidney infection (PD50k) was evaluated for Leptospira interrogans serovars canicola, icterohaemorrhagiae, pomona, and grippotyphosa. All 3 immunogens had similar PD50D values. However, the PD50K values for OE and WC vaccines ranged from 0.05 to 0.80 micrograms (dry weight), whereas the PC vaccines ranged from 7.0 to 14.0 micrograms. Cellular and humoral responses of dogs to serovar canicola WC, OE, and PC vaccines were monitored for 21 weeks. Little difference was observed among the canine humoral responses to the different preparations. Protoplasmic cylinder vaccines sensitized the greater population of lymphoid cells followed by OE and WC. Cross-reactivity was greater in the blastogenic response of lymphoid cell populations than in the humoral response.

During a period of six years 7,555 bovine sera, 421 canine sera, 251 porcine sera and 135 equine sera were tested for agglutinins to Leptospira interrogans serotypes canicola, grippotyphosa, hardjo, icterohemorrhagiae, pomona and sejroe. The bovine sera reacted predominantly with hardjo and/or sejroe at a rate of 15% compared to 3.5% with pomona. Breeding or abortion problems were associated with pomona but not with sejroe/hardjo agglutinins. The canine sera reacted to canicola (9.9%y and icterohemorrhagiae (5.4%), tcted predominantly with canicola (8.9%) and icterohemorrhagiae (8.1%).

A serologic survey was made of the prevalence of common leptospiral infections in horses in California. A total of 465 serums were tested, using the microscopic agglutination method, against 5 leptospiral serotypes: Leptospira pomona, Leptospira icterohaemorrhagiae, Leptospira canicola, Leptospira grippotyphosa, and Leptospira hardjo. Of the serums tested, 127 (27.30%) were positive against 1 or more of the leptospires, with percentage distribution among the reactors as follows: L pomona, 12.47%; L icterohaemorrhagiae, 10.32%; L canicola, 3.22%; L grippotyphosa, 0.86%; and L hardjo, 0.43%. The prevalence of reactors to leptospiral antigens did not differ with the age or sex of the animals.

Antibodies to serotypes representing one or more of 10 Leptospira serogroups were detected in the sera of 1766 (49-1 per cent) cows and heifers out of a total of 3600 sera tested. Infection by the Hebdomadis serogroup was the predominant infection; antibodies to serotype sejroe were found in 1503 (41-8 per cent) sera. Antibodies to the Icterohaemorrhagiae (7-7 per cent) and Balllm (7-3 per cent) serogroups were the next most common, while antibodies to the Australis, Javanica, Canicola, Panama, Cynopteri, Pyrogenes and Autumnalis serogroups were detected in a small number of animals. Only the Hebdomadis and Icterohaemorrhagiae serogroups were associated with clinical disease.

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.