Improved Microtechnique for the Leptospiral Microscopic Agglutination Test

A method for improving the original Galton microtechnique for detecting leptospiral antibodies has been developed. Simultaneous titrations were performed on 281 animal and human sera and 17 hyperimmune sera with the microscopic agglutination (MA) test and the improved microtechnique. Reproducibility of the improved microtechnique was determined independently on 65 animal sera by two laboratory sections. The results obtained by comparing positive test data from human and animal sera indicated that agreement between the original MA test and this new method exceeded 94%, whereas the original Galton microtechnique and the original MA test agreed in a maximum of 77% of the tests. This study indicates that the results obtained with the improved microtechnique are much more comparable to results obtained with the original MA test than are those obtained with the original Galton microtechnique.     

Evaluation of a Hemagglutination Test for Human Leptospirosis

An indirect hemagglutination test for the diagnosis of leptospirosis is described; the test uses a soluble antigen from serotype patoc to sensitize sheep erythrocytes which are then fixed with glutaraldehyde. Evaluation of this procedure indicates that it is more reliable than the conventional macroscopic agglutination test and, in contrast with both microscopic and macroscopic agglutination tests, is positive only with sera from persons with current leptospiral illness. The test is simple and convenient and sensitized fixed cells may be stored for at least a year. In comparison with the macroscopic and microscopic tests, only a single antigen is required.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

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.     

Serological Analysis by Enzyme-Linked Immunosorbent Assay Using Recombinant Antigen LipL32 for the Diagnosis of Swine Leptospirosis

Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14–60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys.     

Microagglutination Procedures for Febrile Agglutination Tests

Febrile agglutination tests were done by using as antigens Brucella abortus, Salmonella group D, Proteus OX19, and Pasteurella tularensis. Comparison of results from 23 sera showed that the microtechnique, rapid slide, and test tube methods gave similar titers, although those from the microtechnique were generally higher. The sensitivity of the microtechnique depended upon the concentration of antigen, and, to obtain reproducible results, the optimal concentration of antigens had to be determined by preliminary titrations against specific, positive control antisera. Readability of reactions in the microtechnique was enhanced by adding the dye Safranin O to diluent for antigen and by use of V-type, rather than U-type, microtiter plates. Tests were also done to determine the effects of dye and salt concentrations, pH, and temperature of incubation upon the titer of agglutinations by the microtechnique. Our results indicated that the microtechnique could be used for agglutination tests involving febrile antigens. The procedure is less time-consuming than the tube method and requires less antigen and serum than the latter method or the rapid slide method.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11 % positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM anti-brucella antibodies.     

Comparative study of cell-immunoenzymatic methods for the estimation of IgG and IgM anti-Brucella antibodies in the diagnosis of human brucellosis

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11% positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM antibrucella antibodies.     

Laboratory diagnosis of leptospirosis

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.     

Evaluation of ELISA for the Detection of Toxoplasma Antibodies in Swine Sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from > 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving < 10 EIU were negative in the other tests, and all those with > 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10–70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Leptospira antibodies in wild boars (<Emphasis Type="Italic">Sus scrofa</Emphasis>) in Slovenia

Serum samples collected from 437 shot wild boars (Sus scrofa) were tested for the presence of antibodies against Leptospira interrogans sensu lato in wild boar in Slovenia. Assessment of leptospira-specific antibodies was performed by microscopic agglutination test. Antibodies against at least one of the pathogenic serovars were detected in 200 (45.8%) sera. From 200 positive samples, 100 samples (50%) had positive titre against a single serovar, while 100 (50%) samples had positive titres against two or more serovars. The most frequently detected antibodies were those against serovar Tarassovi. This investigation confirmed the presence of different pathogenic serovars in wild boar across Slovenia. It can be concluded that wild boars are natural reservoirs of at least some of the leptospiral serovars that represent a potential source of leptospirosis for other wild and domestic animals, as well as for humans.     

Leptospira strains kept at the National Centre for Leptospirosis in Rome, Italy

Since the National Centre for Leptospirosis (Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome) was established in 1956 by B. Babudieri, efforts have been devoted to identifying new Leptospira isolates and maintaining a collection of strains that today comprises 670 strains, 550 of which have been totally or partially classified, and 120 are still under study. This collection includes 23 serogroups and 156 serovars of pathogenic leptospires, and 32 serogroups and 54 serovars of saprophytic leptospires. The conventional serogroup and serovar identification, mainly based on antigenic relatedness, is tedious and time-consuming, requiring the maintenance of a comprehensive collection of serovar reference strains and the preparation of the corresponding rabbit antisera. Although considerable difficulties are encountered in the classification of leptospires at the serogroup and serovar level, this classification system is essential to obtain information on the epidemiology of leptospirosis in the different geographical areas. Serovar identification has become faster with the introduction of pulsed-field gel electrophoresis (PFGE) of large DNA fragments obtained after digestion of leptospiral DNAs with rare-cutting restriction enzymes. This technique has been successfully utilized to discriminate between closely related serovars of the Leptospira interrogans complex. We have recently used PFGE to characterize several Italian leptospiral isolates, confirming that PFGE analysis combined with microscopic agglutination test (MAT) with monoclonal and polyclonal antibodies can be used as an accurate and reliable method to compare and classify leptospires.     

Specificity of anti-human IgG antibodies in antiglobulin (Coombs) sera.

The agglutination test with latex particles coated with aggregated human IgG was introduced into the evaluation of Coombs serum as an additional test for anti-IgG antibody activity. In Coombs sera prepared by the conventional immunization method employing Freund's adjuvant, latex agglutination titers were found much lower than those of anti-D-coated red cell agglutination. On the other hand, in sera prepared by other immunization methods, such as the one according to Haynes and Chaplin (1971), anti-IgG antibody response was readily observed by IgG-coated latex agglutination. Specificity of anti-IgG antibodies in the latter sera seems to be predominantly directed to aggregated human IgG.     

Comparison of antibodies to Leptospira in white-tailed deer (Odocoileus virginianus) and cattle in Ohio

A survey was conducted to determine the prevalence of leptospiral antibodies in sera from 248 white-tailed deer (Odocoileus virginianus) in Ohio. The sera were collected at check stations during the hunting season in 1983. The microscopic agglutination microtiter test was used to determine the presence of antibodies to Leptospira interrogans serovars pomona, icterohemorrhagiae, canicola, hardjo, and grippotyphosa. Eighteen of 248 (7.3%) serum samples had antibody titers (greater than or equal to 1:100) to at least one of the five serovars tested, with three of these samples reacting to more than one serovar. Prevalence did not differ significantly between sex or age groups. The serovar antigens reacting most frequently with serum antibodies were grippotyphosa (10 of 22, 45.5%) and pomona (eight of 22, 36.4%). Sera agglutinating with pomona antigen had higher titers (ranging from 1:200 to 1:6,400) than did sera agglutinating with the other serovars. These results were compared to results obtained from cattle tested at the Ohio Department of Agriculture Laboratories during 1983. There was a significant relationship between pomona infections detected in deer and cattle (P less than 0.05), but not with grippotyphosa.     

Evaluation of the diagnostic potential of recombinant leptospiral OMP A-like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals

Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963 bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36 kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n = 53) and negative (n = 52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.     

Evaluation of Micro Complement Fixation Tests for Antibodies Against Group A Streptococcal M and M-Associated Antigens in Rabbit and Human Sera

The microslide gel-diffusion and macro-tube agglutination techniques to detect Brucella canis antibodies in dogs were compared. Sera from dogs experimentally infected with B. canis and a random sample of dog sera with unknown histories of exposure to this organism were examined. The results of the gel-diffusion method employing specific rough Brucella saline-extract antigens of B. canis and Brucella ovis were comparable to those obtained by the tube agglutination test. The easily prepared, stable R antigen in the freeze-dried form offers a convenient, simple, and reliable diagnostic method for the serological detection of canine brucellosis by the gel-diffusion test.     

Development and evaluation of latex agglutination test coating with recombinant antigen,LipL32 for serodiagnosis of human leptospirosis

Leptospirosis is a widespread zoonotic disease caused by Leptospira interrogans. Symptoms of disease range from mild symptoms to serious complications including, jaundice, pulmonary hemorrhage, renal and hepatic failure, which may prove fatal. Clinical presentations of this disease are similar with other febrile illness. Therefore, rapid and appropriated laboratory diagnostic tests are needed to aid clinical case identification. As these reasons, objective of this study is to develop and evaluate a simple latex agglutination test coating with recombinant leptospiral antigens, LipL32 for serodiagnosis of human leptospirosis. Firstly, lipl32 gene was amplified from genomic DNA of Leptospira interogans serovar Pyrogenes. Then PCR product of lipl32 gene was ligated with pGEX-2T plasmid, generating pGRK32 recombinant plasmid. Recombinant GST-LipL32 protein was overexpressed and subsequently purified by using Glutathione-Agarose Resin. Recombinant GST-Lipl32 protein was coated on latex beads for development latex agglutination test (LAT). The relative sensitivity, specificity and accuracy of the developed LAT were compared with indirect immunofluorescences assay (IFA) for detection of anti-leptospiral antibodies in 30 human leptospirosis samples, 30 healthy blood donor samples, 10 dengue fever positive samples, 10 scrub typhus positive samples, and 10 melioidosis samples. Results showed that the developed LAT showed sensitivity, specificity and accuracy: 66.66%, 86.66%, and 80.00%, respectively, comparing with IFA method. Moreover, Kappa analysis showed agreement rate of the two methods were 0.421. It concluded that our developed gave compatible result with IFA. Additionally, Our LAT are simple, rapid and suitable for detection in the field. However, for better sensitivity, diagnostic specificity, positive predictive value, negative predictive value, accuracy and Cohen’s kappa comparison should be done in larger amounts of sera samples.     

Leptospires in the marine toad (Bufo marinus) on Barbados

Leptospires were isolated from the kidneys of four of 211 toads (Bufo marinus) caught on Barbados. Two of the isolates were identified as Leptospira interrogans serovar bim in the Autumnalis serogroup (the most common cause of leptospiral illness on Barbados), and two as possibly new serovars in the Australis serogroup. Sera from 198 of the toads were examined by the leptospire microscopic agglutination test. Forty-two (21%) were positive at titers of greater than or equal to 1:100, and 54 (27%) at greater than or equal to 1:50. The predominating serogroups were Australis (50%), Autumnalis (23%) and Panama (13%). The agglutination tests on the culture-positive toads showed that serologic studies alone may be of limited value in these animals. Bufo marinus can harbor pathogenic leptospires, and it may be a significant source of the Autumnalis serogroup infections in the Caribbean.     

Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource,hyper-endemic setting with varied microgeographic variation in reactivity

The microscopic agglutination test (MAT) is the standard serological reference test for the diagnosis of leptospirosis, despite being a technically demanding and laborious procedure. The use of a locally optimised MAT panel is considered essential for proper performance and interpretation of results. This paper describes the procedure of selecting such an optimised panel for Sri Lanka, a country hyper-endemic for leptospirosis. MAT was performed using 24 strains on 1132 serum samples collected from patients presenting with acute undifferentiated fever. Of 24 strains, 15 were selected as the optimised panel, while only 11% of serum samples showed positivity. A geographical variation in predominantly reactive serovars was observed, whereas reactivity was low with the saprophytic strain Patoc. Testing with paired sera yielded a higher sensitivity but provided only a retrospective diagnosis. Serological tests based on ELISA with complementary molecular diagnosis using PCR are a feasible and robust alternative approach to diagnose leptospirosis in countries having a higher burden of the disease.     

Microtiter Plate Agglutination Test for Brucella canis Antibodies

A micro-agglutination test for the antibodies to Brucella canis produced similar results to those obtained with the standard tube agglutination method in human and canine sera. The micromethod does provide an economical means of screening sera for the presence of antibodies.     

A comparative evaluation of the sensitivity of different methods for the serological diagnosis of leptospirosis

In this work the comparative evaluation of the sensitivity and serological specificity of the microcapsular agglutination (MCA) test, the passive hemagglutination (PHA) test and the microagglutination (MA) test are presented. In the MCA test leptospiral antigens, adsorbed on synthetic carrier capsules produced by Japan Lyophilization Laboratory, were used and the PHA test was made with the use of polyvalent erythrocyte diagnosticum. The study of blood serum samples from 46 leptospirosis patients revealed that the values of antibody titers in the PHA and MCA tests were 5.5-8.1 times higher than the traditional MA test. In the MCA and PHA tests antileptospiral antibodies could be detected as early as on days 1-3 of the disease when the results of the MA test were negative or very low. The maximum values of antibody titers in the MCA and PHA tests were detected on days 11-15 of the disease and in the MA test, on days 21-25. The MCA and PHA tests are genus-specific and permit the detection of antileptospiral antibodies irrespective of the serogroup of the infective agent. In the study of the blood sera of 40 patients with diseases of nonleptospiral etiology the MCA and MA tests yielded false positive results in 7.5% and the PHA test, in 12.5% of cases in titers below the diagnostic level. These data are indicative of high sensitivity and specificity of the serological tests used in this study.