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Growth, virulence, and immunogenicity of Leptospira interrogans serotype szwajizak.

Leptospira interrogans serotype szwajizak was characterized by (1) its growth in polysorbate 80-bovine albumin medium, (2) its virulence and course of infection in laboratory animals, and (3) its immunogenicity. Growth of this organism was continuous and vigorous at 29 C and 37 C in liquid medium for 10 serial subcultures. Some specific lots of agar were superior to other agars if tested for the ability to support the growth of small inoculums. Individual colonies resulted from growth of small inoculums on solid polysorbate medium. Virulence of the organisms did not appear to be altered by 10 serial subcultures in liquid medium incubated at 29 C. The estimated median lethal dose of szwajizak for hamsters by the intraperitoneal route was 2 cells. Virulence, infectivity, and pathogenicity of szwajizak were shown in the hamster and the guinea pig. Protection results indicate the heat-inactivated szwajizak bacterin was a substantially better immunizing agent than the chemically inactivated bacterin. Serotype hardjo bacterins provided hamsters some protection against death if challenge exposed with szwajizak, but afforded no protection against infection.

Animals

Axial filament involvement in the motility of Leptospira interrogans.

Motility mutants of Leptospira interrogans serovar illini were isolated and analyzed by dark-field and electron microscopy. Mutants were obtained by screening for small colonies after nitrosoguanidine treatment. One class of mutants did not have hook- or spiral-shaped ends. In addition, the axial filaments from these mutants were not coiled. An analysis of revertants of two of the mutants in this class indicated that the mutations were pleiotropic with respect to motility, hook- and spiral-shaped ends, and axial filament coiling. We conclude that the axial filaments and the hook- and spiral-shaped ends are involved in L. interrogans motility.

Cell Wall

Immunochemistry of extracts from Leptospira interrogans serotype hardjo.

Antigens from Leptospira interrogans serotype hardjo grown in modified Korthof's medium were obtained by ethanol and alkaline extraction procedures and their chemical and serological properties were compared. The protein to polysaccharide ratio in the ethanol extract was 1:1-8 and in the alkali extract was 1-7:1. The lipid content of the latter was twice that of the former (8-5%, w/w). There was an inverse relationship of the protein and carbohydrate contents of the two preparations, the total reducing sugar being higher in the ethanol extract whereas protein was higher in the alkali extract. Both preparations contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose and galacturonic acid but in different amounts. No muramic acid or 2-keto-3-deoxyoctonate was detected. Both extracts contained erythrocyte sensitizing substances which, in the passive haemagglutination absorption test, appeared to be closely related antigenically but not identical. In the electron microscope, thin sections of the ethanol extract showed trilaminar outer envelope-like material.

Antigens, Bacterial

The effects of alkalinity and hypertonicity on the morphology and motility of Leptospira interrogans (biflexa) strain B16.

Effects of alkalinity and hypertonicity on the motile behaviour of Leptospira interrogans (biflexa) B16 were observed, quantified, and compared with effects previously shown by similar factors on the motility of eubacteria. Leptospira interrogans tolerated relatively high concentrations of hydroxide ions. Motility similar to that in controls was observed at pH values up to 9-8; but at pH 10-0 motility declined sharply with time of exposure, and there was structural alteration, visible as a blebbing of the cell envelope. Unlike the behaviour of eubacteria, immobilization of L. interrogans induced by hydroxide ions could not be reversed by lowering the pH. It is suggested that by restricting entry of hydroxide ions, the cell envelope protects its motility apparatus from adverse effects. Leptospira interrogans was completely immobilized in 0-5 M and 1-0 M-sucrose solutions. Unlike the eubacteria, leptospires were incapable of spontaneous reversion to motile forms and resumption of motility was dependent on both concentration and time of exposure to sucrose. Deuterium oxide did not affect movement, suggesting that even though leptospire endoflagella and the exoflagella of eubacteria are analogous, the motile behaviour of L. interrogans is significantly different from that of eubacteria.

Culture Media

A serological survey of Leptospira interrogans serotype pomona in Saskatchewan horses.

Leptospira interrogans serotype pomona antibody titres of 1:100 or greater were detected in 12.8% of 408 adult horses from seven of eight sampled herds in Saskatchewan. The geographical distribution of the seropositive horses was widespread throughout the agricultural area of the province. The geographical distribution and the cumulative increase in prevalence with age suggested that serotype pomona is enzootic in the equine population of Saskatchewan.

Animals

Experimentally induced Leptospira interrogans serovar autumnalis infections in young swine.

Leptospira interrogans serovar autumnalis strain 621 was proven to be infective for swine by intranasal and ocular inoculation and by contact exposure. The course of the disease was that of a chronic asymptomatic infection. Throughout the experiment, leptospires were observed in all tissues examined by darkfield microscopy of 10% tissue suspensions and examination by indirect fluorescent antibody technique of frozen tissue sections. Infected pigs had increasing antibody titers, as determined by the microscopic agglutination test. The organism spread from intranasally inoculated pigs to contact controls.

Administration, Intranasal

Use of hamster antisera in the preliminary differentiation of Leptospira interrogans servars hardjo and balcanica.

Preliminary differentiation of Leptospira interrogans serovars hardjo and balcanica can be made with antisera that are collected from hamsters after experimental infection with serovar hardjo and selected for high homologous and low or nil heterologous titres, as measured by the microscopic agglutination test. Rabbit antisera against serovars hardjo and balcanica, cattle antisera against hardjo and sheep antisera against the serogroup Hebdomadis each agglutinated hardjo and balcanica to a similar titre.

Agglutination Tests

Experimental infection of calves and sheep with Leptospira interrogans serovar balcanica.

Two of four calves inoculated with Leptospira interrogans serovar balcanica developed low microscopic agglutinating (MA) titres to serovar hardjo. A third calf had an MA titre of 1:1024 by day 19 post-inoculation (PI). Transient leptospiruria was recorded in one calf on days 12 and 13 PI. An in-contact calf did not seroconvert. None of the calves had fever or other clinical signs of disease. Four ewes inoculated with balcanica developed MA titres to hardjo by day 13 PI, and a transient leptospiruria between days 14 and 25 PI. None of the ewes showed any evidence of clinical disease and three of them delivered healthy lambs 22 to 64 days PI. One ewe had mild lesions of focal interstitial nephritis.

Agglutination Tests

Survival of Leptospira interrogans serovar pomona in an acidic soil under simulated New Zealand field conditions.

Leptospira interrogans serovar pomona was found to survive for at least 42 days in a typical New Zealand soil under simulated winter field conditions. The soil was markedly acidic with a pH of 5.5 and survival times were not reduced even when its water content was only 23%. The values of both these parameters are considerably less than previously recorded for the survival of leptospires in soil. Two methods were used to recover leptospires from the soil microflora. One was the culture of a membrane filtrate in EMJH media with or without contaminant-suppressing additives and the other was the direct inoculation of soil-washings into hamsters. Both techniques proved to equally sensitive. It was estimated that following the addition of 5 X 10(8) leptospires to the soil samples less than 2 X 10(4) were present after six weeks.

Animals

Nature of antigenic determinant of serovar-specific antigen of Leptospira interrogans serovar hebdomadis.

The nondialyzable delipidized serovar-specific main antigen (NDTM antigen) of Leptospira interrogans serovar hebdomadis strain Hebdomadis (a variant which can grow in a synthetic medium) showed a strong inhibition of the complement fixation between the serovar-specific main (TM) antigen of this strain and the homologous antiserum. The inhibitory effect of the NDTM antigen was completely lost by treating the antigen with proteolytic enzymes, and the fractions of TM antigen containing amino sugar, neutral sugars, and lipids did not show any inhibition of complement fixation, indicating that the antigenic determinant of this strain is related to proteins. NDTM antigen contained more hydrophilic amino acids than hydrophobic amino acids, whereas TM antigen contained more hydrophobic amino acids than hydrophilic amino acids. The amino acid compositions of NDTM antigens of hebdomadis strain Hebdomadis (variant) and kremastos strain Kyoto, which belonged to the same serogroup, were considerably similar. Difference was found in the amounts of methionine, arginine, lysine and glutamine acid.

Amino Acids

Experimental infection of calves with Leptospira interrogans serotype szwajizak.

This is a report on the experimental infection of cattle with Leptospira interrogans serotype szwajizak. The principal clinical features in three 4-week-old Holstein-Friesian heifers intravenously inoculated were fever, listlessness, anorexia, weakness, and diarrhea. Szwajizak was isolated from the blood for the first 5 to 8 days after inoculation. Leptospires were recovered from kidneys, but not from liver, spleen, brain, or urine. Two of the 3 calves produced homologous agglutinins, with maximum serum titers of 1:80 and 1:160. The sera of the 2 calves that developed szwajizak agglutinins showed cross reactivity with wolffi and hardjo antigens. There were not significant differences of plasma and urine constituents between the inoculated calves and the control calf. Endocardial hemorrhages and large soft black-red spleen were present in the 2 calves given the greatest numbers of leptospires. Histopathologically, kidney of all infected calves had multiple small foci of lymphoid cells, chiefly pasmacytes, which were present in periglomerular and interstitial areas.

Animals

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans.

Culture Media

Experimental infections of brush-tailed possums, common wombats and water rats with Leptospira interrogans serovars balcanica and hardjo.

Of 12 brush-tailed possums (Trichosurus vulpecula) inoculated with Leptospira interrogans serovar balcanica 11 developed migroagglutination (MA) antibody to jardjo antigen by 14 days postincubation (PI). Leptospiruria was observed in 2 possums 117 to 145 days PI. Of 6 possums inoculated with serovar hardjo 4 developed low short-lived titres by day 18 PI. Two of 3 wombats (Vombatus ursinus) inoculated with balcanica had high MA titres (greater than or equal to 1:128) by day 16 PI and leptospiruria occurred by day 16. One wombat inoculated with hardjo developed a low MA titre. Low transitory MA titres to hardjo were found in 1 of 3 water rats (Hydromys chrysogaster) after inoculation with balcanica and 1 of 2 given hardjo. Histopathological examination of kidneys revealed mild to moderately severe focal interstitial nephritis in 4 of 8 possums, in 2 wombats and in 2 water rats following experimental infection with balcanica. Similar lesions were observed in 2 of 4 possums, 1 wombat and 2 water rats following experimental infection with hardjo.

Agglutination Tests

Viability of Leptospira interrogans serotype grippotyphosa in swine urine and blood.

Normal swine urine, devoid of its microbial flora, diminished the viability and virulence of Leptospira interrogans serotype grippotyphosa. Bovine serum albumin diluent effectively offset the urine's deleterious effects. Membrane filtration (0.45 micrometer) rendered urine free of bacteria, but permitted passage of limited numbers of leptospires. Most of the commonly used anticoagulants did not alter viability of grippotyphosa in whole blood. However, EDTA significantly reduced the number of viable cells.

Animals

'Viscotaxis', a new behavioural response of Leptospira interrogans (biflexa) strain B16.

When in a non-viscous environment and confronted with a viscous one, Leptospira interrogans (biflexa) strain B16 preferentially selected the latter. We have designated this positive response to a viscosity gradient as 'viscotaxis'. Using an originally designed experimental chamber, a pool of leptospires were faced with capillary tubes containing either polyvinylpyrrolidone (PVP) or N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES). Leptospires suspended in HEPES responded positively to 2% (w/v) PVP in capillaries, and migrated into them in large numbers in 1 h. No response was observed when the chamber and capillary tubes contained solutions of the same viscosity. As the viscosity of PVP was increased, a proportionally larger number of leptospires migrated into it. This newly observed aspect of leptospiral behaviour may have ecological significance.

Culture Media

The serologic and cultural prevalence of Leptospira interrogans serovar balcanica in possums (Trichosurus vulpecula) in New Zealand.

In a serologic and cultural survey of 127 brush-tailed possums (Trichosurus vulpecula) occupying pasture land in New Zealand, leptospires of the Hebdomadis serogroup were obtained from 48 (38%) of the animals sampled. Eight isolates were identified by cross-absorption agglutin ation studies as being Leptospira interrogans serovar balcanica. There was a marked age difference in prevalence with 41 positive cultures from 64 mature adults (64%) and no recoveries being made from juveniles. Isolation of leptospires was aided by the use of a new technique involving the homogenation of whole kidneys in gamma sterilized plastic bags in a "Coleworth Stomacher". The use of this apparatus allowed the processing of whole kidneys and the technique was efficient in both the recovery of leptospires and the prevention of contamination. In view of the fact that serovar balcanica has been recorded previously only in East Europe in man, cattle and pigs, the high prevalence of infection in a wild animal population in New Zealand is an interesting development in the world distribution of this serovar.

Animals