Serodiagnosis of canine leptospirosis by solid-phase enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Leptospira interrogans serotype canicola in dogs was developed and evaluated. Comparison of the ELISA with the microscopic agglutination test (MAT) showed that, during the first two weeks after an experimental infection with serotype canicola, the ELISA detected antibody at higher dilutions than the MAT. After the second week post-infection both tests detected antibody at almost equal titres (r = 0.89). The outer envelope (OE) antigen of serotypes icterohaemorrhagiae, copenhageni and canicola was fairly serotype-specific, whereas the pellet (P) antigen showed more cross-reactivity. Both OE and P antigen of Leptospira biflexa strain Patoc I could be used as cross-reacting antigen in the ELISA. Compared to the MAT, the ELISA has some technical advantages. It is suggested that the ELISA would be useful as a screening test.     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of Taenia saginata cysticercosis in cattle.

Serum IgG response of cattle with cysticercosis caused by Taenia saginata was studied in an enzyme-linked immunosorbent assay (ELISA) where a T. saginata metacestode surface extract was used as antigen. In experimentally infected calves, a sharp rise in specific antibody levels was found 3-4 weeks after the infection followed by a logical level of detection corresponded to about 25 cysts. The ELISA was employed in cattle herds where cysticercosis outbreaks had occurred and also in supposedly uninfected herds. Significantly increased antibody levels were found in the herds with massive cysticercosis cases. The test was not adapted for individual diagnosis as some animals of the uninfected herds, especially within the older age groups, had elevated antibody values. The ELISA was, however, useful in the investigation of outbreaks to determine the extent and pattern of the infection in the herd. The rate of decline in antibody levels in these herds was studied by follow up sampling. The increased antibody levels in the infected herds were also reflected in colostrum-fed calves. This observation was employed to estimate the time of infection.     

Latest developments in the enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISAs) for the serologic detection of both antigen and antibody in monitoring programs of commercial poultry flocks have begun to be recognized as an improvement over more conventional diagnostic procedures. The feasibility of employing double-antibody sandwich assays for the detection of virus without prior isolation of virus has been demonstrated and shows promise as the method of choice for the detection of lymphoid leukosis virus shedding. The versatility of indirect ELISA for the measurement of antibody induced by a wide variety of potential pathogens using a single basic overlapping ELISA system has also been demonstrated. It shows potential as a likely candidate to replace some of the more costly and time-consuming or less sensitive conventional serologic methods that do not overlap. Although some aspects of the two major types of immunoassays currently used in poultry health may need some modifications or improvements before delivery for routine use, it is likely that the use of computer-assisted ELISA will gain increased acceptance and use as the preferred way to efficiently and accurately monitor the health of poultry flocks on a broad scale.     

Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory- secretory antigen

An excretory-secretory (ES) antigen was used in a serodiagnostic enzyme-linke immunosorbent assay (ELISA) for swine trichinosis. ELISA procedures included a double- antibody test, using either an anti-swine IgG or a protein A enzyme conjugate, and a triple-antibody test using a pig IgG heavy-chain specific second antibody with a conjugated third antibody. The ES antigen was effective in eliminating all false-positive reactivity in sera from farm-raised hogs. The triple-antibody procedure was more sensitive and demonstrated a greater efficiency in detecting positive animals and early seroconversions. Naturally-infected pigs with worm burdens as low as 0.01 larvae per gram (LPG) of diaphragm were seropositive using these procedures. Seroconversion in experimentally-infected animals receiving low doses of muscle larvae (500) occurred considerably later than in animals receiving high doses (10000). This might account for false-negative reactions in naturally-infected animals with very low (less than 0.1 LPG) worm burdens.     

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Serodiagnosis of bovine paratuberculosis by use of a dot enzyme-linked immunosorbent assay

A dot ELISA was developed for detection of antibodies to Mycobacterium paratuberculosis. The assay was evaluated by testing sera from cattle that were determined, by bacteriologic culturing of feces, to be infected with M paratuberculosis and were suspected of having clinical disease. Further evaluation involved testing sera from cattle in which M paratuberculosis had not been isolated from feces on several attempts. Results of the dot ELISA were positive for sera from 86 of 101 infected cattle, and results were negative for sera from 64 of 64 noninfected cattle. Results of conventional ELISA and agar gel immunodiffusion (AGID) tests were positive for 79 of 99 and for 51 of 101 infected cattle, respectively. The dot ELISA also was evaluated by comparing results of testing 708 sera with results of bacteriologic culturing of matched fecal samples from 262 cattle in 3 central Ohio dairy herds known to include cattle infected with M paratuberculosis. Results of the dot ELISA were positive for 25 of 39 sera from cattle with positive results on culturing of concurrently obtained fecal specimens. The dot ELISA results were negative for 661 of 669 sera from cattle with negative results to culturing of concurrently obtained fecal specimens. The 39 sera from cattle with positive results on bacteriologic culturing of matched fecal specimens had positive results for ELISA and the AGID test 25 and 14 times, respectively. The 669 sera from cattle with concurrently negative results on bacteriologic culturing of feces had negative results to ELISA and the AGID test 559 and 668 times, respectively.     

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.     

An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus.

The application of the indirect enzyme-linked immunosorbent assay (ELISA) for detection of infectious bursal disease virus antibodies in chicken serum was investigated. The test procedure involved the coating of concentrated infectious bursal disease virus antigen onto polystyrene tubes, followed by the addition of chicken anti-infectious bursal disease virus serum and horseradish peroxidase labeled rabbit anti-chicken globulin. As an indicator substrate, 5-aminosalicylic acid, with the oxidant H2O2 was added. The reaction was stopped by 3M NaOH and the colour intensity of the reaction mixtures read in a spectrophotometer at 449 nm. The ELISA test was found to be a precise, sensitive and reproducible means of measuring infectious bursal disease virus antibodies in chicken and turkey sera.     

Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA)

Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.     

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of dourine

The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.