Enzymatic radioimmunoassay for detecting Leptospirainterrogans serovar Pomona in the urine of experimentally-infected pigs

doi:10.1016/0378-1135(85)90053-7

Veterinary Microbiology

Volume 10, Issue 3, April 1985, Pages 279-286

https://doi.org/10.1016/0378-1135(85)90053-7 Get rights and content

Abstract

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.

References (13)

B. Adler et al.
The sensitivities of different immunoassays for detecting leptospiral antigen
Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. I. Orig. Reihe A
(1982)
S.A. Ballard et al.
The immunoglobulin response of swine following experimental infection with Leptospira interrogans serovar pomona
Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. I. Orig. Reihe A
(1984)
B. Adler et al.
Serological and protective-antibody responses of rabbits to leptospiral antigens
J. Med. Microbiol.
(1978)
B.W. Christmas et al.
Dairy farm fever in New Zealand—isolation of L. pomona and L. hardjo from a local outbreak
N.Z. Med. J.
(1974)
D.L. Coffin et al.
Detection of leptospires by fluorescent antibody
Am. J. Vet. Res.
(1962)
C.C. Harris et al.
Ultrasensitive enzymatic radioimmunoassay: Application to detection of cholera toxin and rotavirus

There are more references available in the full text version of this article.

Cited by (9)

Moving past serology: Diagnostic options without serum
2016, Veterinary Journal
Citation Excerpt :
Urine has been used as a medium for testing animal diseases and is particularly useful to test for Leptospira infection. Increasingly, PCR is used as the method of choice for the detection of the organism (Chappel et al., 1985; Fang et al., 2014; Hamond et al., 2014). Urine testing is also used to detect if performance enhancing drugs have been used in horses (and humans athletes; Thevis et al., 2016).
Detecting antibodies formed in serum in response to infection is the traditional function of serology. Diagnostic modalities have included complement fixation tests, agar gel immune-diffusion, radioimmunoassay, ELISA and immunofluorescence. More recent technology now allows for the direct detection of pathogens by PCR. This review details the options for diagnostic testing using specimen types other than serum, identifying the advantages and disadvantages of these options and providing evidence for more widespread use of these techniques and specimen types.
Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine
1998, Research in Veterinary Science
A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10³ to 10² leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.
Magnetic immuno capture PCR assay (MIPA): Detection of Leptospira borgpetersenii serovar hardjo
1997, Veterinary Microbiology
Magnetic immuno PCR assay (MIPA) was developed for the rapid detection of leptospires excreted in urine samples (n = 59) collected from 35 experimentally infected cattle. The immunomagnetic separation of leptospires from inhibitors in frozen formalin fixed bovine urine prior to PCR detection resulted in a marked improvement on previous detection methods. MIPA is a rapid 5 step protocol requiring 70 mins preparation time prior to amplification, which consistently detects 10¹ organisms. MIPA detected 76% (38/50) of culture positive urines and in addition three urines that were culture negative were shown to be positive by this method of detection. Consequently we conclude that whilst MIPA is an improvement on previously published PCR detection methods, the culture of the organism is still the standard against which other detection methods have to be compared.
Comparison of diagnostic procedures for porcine leptospirosis
1992, Veterinary Microbiology
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 (⩾ 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.
Detection of leptospires in biological fluids using DNA hybridisation
1987, Veterinary Microbiology
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-μl spot on nylon membranes was 160 pg of leptospiral DNA or 1.1 × 10³ 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°C.
Experimental trials on the use of radioimmunoassay for the detection of leptospiral antigens in urine
1986, Veterinary Microbiology
Trials were conducted on the use of the solid phase radioimmunoassay (RIA) to detect leptospires or their antigens in simulated urine samples. The procedure was relatively simple to perform and appeared to be specific in detecting certain numbers of leptospiral organisms or their antigens in experimentally prepared samples. With this technique, it was possible to examine individual or pooled urine samples for the presence of leptospires within half a day. This technique may be of value for the detection of leptospiruric animals if the sensitivity of the technique could be further increased. Suggestions for the improvement of the procedure are discussed.

View all citing articles on Scopus

View full text

Article preview

Veterinary Microbiology

Abstract

References (13)

The sensitivities of different immunoassays for detecting leptospiral antigen

Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. I. Orig. Reihe A

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

Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg., Abt. I. Orig. Reihe A

Serological and protective-antibody responses of rabbits to leptospiral antigens

J. Med. Microbiol.

Dairy farm fever in New Zealand—isolation of L. pomona and L. hardjo from a local outbreak

N.Z. Med. J.

Detection of leptospires by fluorescent antibody

Am. J. Vet. Res.

Ultrasensitive enzymatic radioimmunoassay: Application to detection of cholera toxin and rotavirus

Cited by (9)

Moving past serology: Diagnostic options without serum

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine

Magnetic immuno capture PCR assay (MIPA): Detection of Leptospira borgpetersenii serovar hardjo

Comparison of diagnostic procedures for porcine leptospirosis

Detection of leptospires in biological fluids using DNA hybridisation

Experimental trials on the use of radioimmunoassay for the detection of leptospiral antigens in urine