Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of leptospirosis.

A proficiency testing scheme for the leptospirosis microscopic agglutination test was provided to 37 laboratories in 23 countries in 2002 (round 1) and to 60 laboratories in 34 countries in 2003 (round 2). Thirty-four laboratories participated in both rounds. Each panel consisted of five rabbit serum samples, four of which were antisera raised against pathogenic serovars of Leptospira. One of these samples was a mixture of two different antisera. The rates of false-negative results, calculated on the basis of the assumption that serovars within a serogroup will cross-react, were 11% for round 1 and 14% for round 2. There were regional differences in the rates of false-negative results. The titers reported by laboratories testing for the same sample with the same serovar varied widely. Laboratories that had previously participated in round 1 reported fewer false-negative results in round 2 than new participants (10 and 21%, respectively [P = 0.002]) and reported 0.56 false-negative results per participant, whereas new participants reported 1.23 false-negative results per participant (P = 0.041). Laboratories that had previously participated also reported fewer false-negative results in round 2 than in round 1 when samples common to both rounds were tested (5 and 15%, respectively [P = 0.028]). The titers reported by the new participants were, on average, lower than those reported by the laboratories that had participated previously (P = 0.019) and were significantly more variable (P = 0.001). Analysis of these results suggests a positive impact of proficiency testing on the testing performance of the participating laboratories.

Serological titres to Leptospira fainei serovar hurstbridge in human sera in Australia.

A set of 723 diagnostic sera from human patients, submitted for the microscopic agglutination test (MAT) for antibodies to a group of 6 leptospiral serovars, was also tested by MAT for antibodies to the recently-discovered Leptospira fainei serovar hurstbridge. MAT titres of > or = 128 to serovar hurstbridge were detected in 13.4% of these sera, and titres of > or = 512 in 7.2%. In contrast, none of 62 sera obtained from a control population of laboratory staff gave titres of > or = 128. The difference between the number of titres of > or = 128 given by the two groups of sera was highly significant (P < 0.01). The titres observed may have been due to cross-reactions with other leptospiral serovars, but this could not be demonstrated. An alternative explanation is that serovar hurstbridge is present in the human population.

Prevalence and geographic origin of pigs with serological evidence of infection with Leptospira interrogans serovar pomona slaughtered in abattoirs in Victoria, Australia.

A set of 10,440 sera was collected from pigs slaughtered at Victorian abattoirs. These sera were subjected to the microscopic agglutination test for antibodies to Leptospira interrogans serovar pomona. Identification of the herd of origin was possible for 6511 pigs, and these were derived from 167 herds in Victoria (84% of sera), from 32 herds in New South Wales (8% of sera) and 29 herds in South Australia (8% of sera). The overall prevalence of titres of 512 and above was 3.7%. This was higher (5.3%) among pigs for which the property of origin was unknown than among pigs with identified properties of origin. Among the latter the prevalence was 2.7% (Victoria 0.6%, New South Wales 1.3%, South Australia 25.2%.) Most of the pigs with unknown properties of origin were derived from market groups and were probably typically from smaller herds. Within Victoria a comparison of results with the known pig populations of the 12 statistical divisions indicated that infection was spread throughout the State. Of the 228 identified herds of origin sampled, 32 (14%) had at least one pig with a high titre. However, this may underestimate the proportion of infected herds, as in many cases only a few serum samples were obtained. Of 73 herds from which 25 or more serum samples were obtained, serological evidence of infection was obtained in 18 herds (25%).

Leptospira fainei sp. nov., isolated from pigs in Australia.

Pathogenic leptospires can be causative agents of reproductive problems in pigs. Cultures of uteri and kidneys from two pigs herds in New South Wales and Victoria (Australia) yielded five strains identified as Leptospira on morphological and cultural grounds. Phenotypic characteristics (growth at 13 and 30 degrees C, growth in the presence of 8-azaguanine) were intermediate between those of pathogenic and saprophytic leptospires. No cross-agglutination was observed with reference antisera representing the 24 pathogenic serogroups and the main saprophytic ones. Antiserum against one of the strains did not agglutinate reference stains representative of any serogroup. This provided evidence of a new serovar, designated hurstbridge. Genomic characterization of the five strains was achieved using five molecular approaches. Mapped restriction site polymorphisms in the rrs (16S rRNA) gene were not related to those of any reference strains. Arbitrarily primed PCR fingerprints suggested clonality of the five strains. The strains all showed an identical and unique PFGE profile. PCR, using primers specific for the rrs gene of pathologic leptospires, amplified corresponding sequences from the strains. DNA-DNA hybridization (and reciprocal experiments) using the S1 nucleas/TCA method was performed between one of the strains and the reference strains of Leptospira species. The homology ranged from 0 to 36% (the latter being was Leptospira inadai) thus satisfying the criterion of a new species, Leptospira fainei (type strain BUT 6T). Phylogenetic analysis of 16S rRNA sequence showed that L. fainei and L. inadai formed a clade separate from the previously recognized 'saprophyte' and 'pathogen' clades.

Construction of a shuttle vector for use between Pasteurella multocida and Escherichia coli.

The isolation of a small plasmid from Pasteurella multocida has enabled the construction of a shuttle vector for use between P. multocida and Escherichia coli. The vector pBAC64 contains the origin of replication from P. multocida, an antibiotic resistance gene which functions in P. multocida, and the E. coli vector pUC18. The presence of the pUC18 multiple cloning site together with the lacZ' gene provides a screening method and allows cloning and manipulation in E. coli as well as cloning in P. multocida.

[Isolated lesion of the accessory nerve]. / De geïsoleerde N. accessorius-laesie.

An isolated accessory nerve lesion was diagnosed in three patients. At clinical investigation of patients with this lesion, paresis of the trapezius muscle is found. This finding can be substantiated by electromyography. An accessory nerve lesion is usually caused by trauma (including surgical trauma) or space-occupying lesions such as tumour or abscess. There are also idiopathic forms. The prognosis is poor. Treatment may include electrostimulation, administration of NSAIDs, nerve transplantation and muscle transposition.

Comparison of diagnostic procedures for porcine leptospirosis.

Kidneys and matched serum samples were obtained from 368 pigs slaughtered at three Victorian abattoirs, and originating from 42 farms. Macroscopic lesions (white spots) were observed on 102 of the kidneys. Serum samples were tested by the microscopic agglutination test (MAT) and by an IgM enzyme immunoassay (EIA). Kidneys were cultured for leptospires, examined histologically after Warthin-Starry silver staining and after immunogold silver staining (IGSS), and tested for leptospiral DNA by DNA hybridization. Forty-four infected pigs were identified by culture or immunogold silver staining of kidneys or by high MAT titres (greater than or equal to 1024). Infection was demonstrated in 7.5% of visibly normal kidneys, in 23.5% of kidneys with white spots, and in 48% of kidneys with large white spots, of 1 cm diameter or greater. The apparent (maximum) sensitivities of diagnostic procedures for detecting infection were as follows: MAT (at a titre of either 64 or 1024) 95%; IgM EIA 82%; culture 61%; presence of white spots 55%; IGSS 52%; presence of large white spots 30%; Warthin-Starry silver staining 20%. IGSS, Warthin-Starry staining and DNA hybridization all appeared to be highly specific. Of 22 kidney sections identified as positive by IGSS, 13 showed intact leptospires, and these kidneys were all culture-positive. Nine others showed leptospiral antigen in the kidney tubules but no intact leptospires. Only five of these kidneys were culture-positive.

Leptospira interrogans serovar hardjo is not a major cause of bovine abortion in Victoria.

The aim of this study was to determine whether evidence could be obtained of foetal infection with Leptospira interrogans serovar hardjo in aborted foetuses collected from dairy farms. Material from 197 abortions occurring over a wide area of Victoria was collected over 3 years. None of 195 foetal kidney cultures or 7 cultures from membranes was positive for leptospiral organisms. Immunogold silver staining for leptospires was performed on sections of kidneys, lungs or heart from 156 foetuses, with negative results. Evidence of transient leptospiral infection in 11 of 123 foetuses was obtained by foetal heart blood serology. Two isolates of L. interrogans serovar hardjo were obtained from the urine of milking cows. These strains were examined by restriction endonuclease analysis and both were shown to be of the genotype Hardjobovis, as have been all Australian isolates studied so far. It appears that foetal infection with serovar hardjo is not associated with any substantial proportion of bovine abortions in Victoria, in contrast to the situation in Northern Ireland. The apparent absence from Victoria of the pathogenic genotype Hardjoprajitno is a possible explanation.

Detection of leptospires in pig kidney using DNA hybridisation.

DNA hybridisation detected leptospiral organisms in homogenised kidneys from experimentally infected pigs, and in homogenates of pig kidneys collected at abattoirs. The technique is easy to perform and had some advantages over cultural and histological methods, in permitting the rapid survey of many kidneys simultaneously. Leptospires added to a homogenate of uninfected kidney could be detected at 10(2) organisms ml-1 by DNA hybridisation, but the technique appeared to be less sensitive than culture.

Effect of maternal vaccination on the susceptibility of growing pigs to leptospiral infection.

Serum samples were collected from 30 piglets, derived from 17 litters, whose dams had been vaccinated against leptospirosis. Microscopic agglutination test (MAT) titres against Leptospira interrogans serovar pomona varied greatly from pig to pig; there was less variation among littermates. Titres declined between 4 and 10 weeks of age, with an uncorrected half-life of 15.5 days, consistent with IgG being the main antibody class involved. Twelve pigs, 4 derived from unvaccinated sows and 8 from sows vaccinated against leptospirosis, were challenged intravenously at 8 weeks of age with leptospires of serovar pomona. Colostrum-derived antibody protected 4 out of 8 pigs, and in 1 of the remaining 4 the serological response was reduced. Three of the protected pigs showed reduced serological responses and in the fourth the response was strong, but delayed. All of the pigs derived from unvaccinated sows developed leptospiraemia and leptospiruria and showed strong serological responses. Protection by colostrum-derived antibody bore an inexact relationship to MAT titre, but a titre of 16 appeared to be sufficient for protection.

Detection of leptospires in biological fluids using DNA hybridisation.

DNA extracted from Leptospira interrogans serovar pomona was labelled with phosphorus-32 by nick translation and used as a genomic probe to detect leptospiral DNA. The sensitivity of detection in a 10-microliter spot on nylon membranes was 160 pg of leptospiral DNA or 1.1 X 10(3) leptospires and assays with nylon membranes were somewhat more sensitive than assays with nitrocellulose membranes. The probe reacted with the pathogenic hardjo and tarassovi leptospiral serovars, but not with other genera of bacteria. To detect leptospires in body fluids, these were treated to free leptospiral DNA and then concentrated on membranes using a Bio-Dot apparatus. Neither serum nor urine interfered with the assay system. The DNA of leptospires added to pig urine was stable for at least 2 h at room temperature and for at least 20 h at -20 degrees C.

Immunogold silver staining for visualization of leptospires in histologic sections.

An immunogold silver stain for leptospires in sections of Formalin-fixed, paraffin-embedded tissues is described. Leptospires were intensely stained, and nonspecific staining of the tissue background was negligible.

Development of an improved selective medium for isolation of leptospires from clinical material.

Standard albumin-Tween 80 medium (EMJH) for growth of leptospires was modified by the addition of six antibiotics to produce a superior, selective medium for primary isolation of leptospires of serovars hardjo and pomona of Leptospira interrogans from clinical material.

Enzymatic radioimmunoassay for detecting Leptospira interrogans serovar pomona in the urine of experimentally-infected pigs.

An enzymatic radioimmunoassay (ERIA) has been developed for detecting Leptospira interrogans serovar pomona in porcine urine. Four grower pigs were experimentally infected with serovar pomona. A total of 39 urine samples was collected, and ERIA was compared with dark ground microscopy (DGM) and culture for demonstrating leptospiruria. Of 20 samples positive by at least one technique, leptospires were detected by ERIA in 14, by culture in 16 and by DGM in 13. ERIA, unlike the other 2 methods, was suitable for use with urine which had been stored frozen for several months.

The immunoglobulin response of swine following experimental infection with Leptospira interrogans serovar pomona.

The antibody response of pigs following experimental infection with Leptospira interrogans serovar pomona was examined using enzyme immunoassay (EIA) and the microscopic agglutination test (MAT). Leptospires elicited the production of both IgM and IgG classes of antibody, with IgG levels persisting for much longer than IgM. A comparison of MAT and EIA indicated that the detection of specific IgM by EIA was potentially useful in distinguishing between past and recent infection in pigs. Agglutinins were also detected in the urine of infected animals but these antibodies could not be detected by EIA.

Comparison of radioimmunoassay with the complement fixation test and the indirect haemolysis test in the field diagnosis of bovine brucellosis.

Sera were collected from female cattle in 118 commercial herds being subjected to a programme to eradicate brucellosis by test and slaughter, in an area in which vaccination of heifer calves with Brucella abortus strain 19 was compulsory. Of 4583 sera positive by the Rose Bengal plate test, the brucellosis radioimmunoassay was positive for 1524, the complement fixation test for 1363 and the indirect haemolysis test for 1141. These figures, and supporting evidence from the eradication programme, suggest that the radioimmunoassay may be a useful supplementary test in problem herds.