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.

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.

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.

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.

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%).

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.

A possible role of Cu2+ ions in bovine antibody-antigen interactions.

A preparation of purified bovine IgG1 was found to contain 0.5 moles of Cu2+ ions per mole of protein. Scatchard analysis of the binding of Cu2+ ions to bovine IgG1 indicated the presence of 4 +/- 1 binding sites with an affinity of 2 x 10(5) M-1. Calculations indicate that in bovine plasma, IgG1 will not compete significantly with albumin for Cu2, whereas a proportion of Cu2+ may well be bound to IgG1 in colostrum. Physiological levels of Cu2+ were found to enhance the interaction between IgG1 antibody and Brucella abortus in the presence of albumin. Irrespective of whether IgG1 binds Cu2+ in vivo, it is possible that Cu2+ can influence the results of serological tests.

The effects of IgG2 and of antigen concentration on prozoning in the complement fixation test for bovine brucellosis.

Addition of Brucella-specific IgG2 to solutions of Brucella-specific IgG1 initially induced prozoning and at higher concentrations prevented all reaction in the complement fixation test (CFT) for bovine brucellosis. Some infected cattle may be diagnosed as brucellosis-free due to a high ratio of specific IgG2 to IgG1. Increasing the concentration of antigen in the CFT reduced the tendency to prozone.

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.

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.

[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.

A comparison of the results of the brucellosis radioimmunoassay and other serological test in experimentally infected cattle.

The serology of 27 heifers found to be positive to culture after inoculation with Brucella abortus strain 544, was studied. Eighteen heifers had previously been vaccinated with strain 19 or strain 45/20 and nine were unvaccinated. Post-infection serum samples were tested for Brucella antibodies by radioimmunoassay (RIA), complement fixation test (CFT), indirect haemolysis test (IHLT) and Rose Bengal plate test (RBPT). All of the unvaccinated heifers showed strong humoral responses to experimental infection in the RIA, CFT, IHLT and RBPT. The CFT and RBPT became positive sooner after infection than the other tests in the unvaccinated heifers. However, in vaccinated heifers the RIA was the most sensitive test early in infection and the results of the RBPT were variable. Three of the vaccinated heifers showed weak and inconsistent humoral responses and, in these animals, the RIA gave fewer false negative reactions than the other tests.

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.

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.

The sensitivities of different immunoassays for detecting leptospiral antigen.

The detection of leptospiral antigen in biological fluids is important for the diagnosis of leptospirosis in animals and man. However the sensitivity of dark field microscopy, the usual detection method, is often inadequate. A comparison was made between the sensitivities of several immunological techniques for detecting Leptospira interrogans serovar hardjo. By staphylococcal coagglutination 10(8) leptospires per ml could be detected and by countercurrent immunoelectrophoresis 10(7) per ml. The best sensitivity obtained by enzyme-linked immunosorbent assay was 10(5) leptospires per ml, and by radioimmunoassay 10(4) to 10(5) per ml. Radioimmunoassay offers the prospect of improved diagnosis of leptospirosis through the detection of leptospiral antigen.

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.

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.