Assessment of two assays for diagnosis of leptospirosis in clinical veterinary samples

Jamie M Fink, Purdue University

Abstract

Leptospirosis is a zoonotic disease caused by pathogenic species of spirochetes of the genus Leptospira. Contaminated urine or water contaminated with urine shed from a carrier play a major role in transmission of the spirochetes among susceptible hosts. In humans the disease is often associated with occupations where there is contact with contaminated water, such as rice paddy or sewage workers. The major symptom most commonly associated with livestock is abortion, but in dogs and humans renal and liver failure occurs in the most severe form of the disease. Antibiotics are used for treatment of leptospirosis, but vaccination is heavily relied upon to prevent disease in some animals. There are currently no approved vaccines licensed for human use in the United States. Animal vaccines often consist of Leptospira serovars that preferentially cause infections of a host and are widely prevalent in the geographical area. Leptospirosis is very difficult to diagnose in both humans and animals because most symptoms are commonly associated with a variety of different diseases. Microscopy can be used for diagnosis, but bacteria are very difficult to find due to their thin, spiral shape and because of the low and inconsistent presence of the organisms in the body fluids. Culture isolation can also be used, but Leptospira spp. growth can take weeks, making this form of diagnosis extremely inefficient. The microscopic agglutination test (MAT) is considered by many to be the "gold standard" in diagnosis of leptospirosis. Unfortunately, MAT can be unreliable since previous vaccination or exposure has the possibility of presenting false positive results. Other serological tests have also been developed, including enzyme-linked immunosorbent assay (ELISA) using various Leptospira spp. antigens, dipstick ELISA, and latex agglutination test, all of which can yield false positive results much like MAT. More recently, molecular diagnostic tests have been developed, but again there are some disadvantages. The objective of this study is to validate a real time PCR for diagnosis of leptospirosis in dogs, as well as to identify an antigen that could be used for detecting the presence of specific IgG in serum of animals exposed to pathogenic Leptospira spp. Both the real time PCR and ELISA were based on a lipoprotein that is highly conserved and unique to pathogenic Leptospira spp., named lipoprotein 32 (LipL32). LipL32 is present in the outer membrane of pathogenic Leptospira, and is known to be expressed upon infection of mammalian hosts. The fact that LipL32 is only present in pathogenic Leptospira spp., is associated with the outer membrane, and is known to be expressed during infection makes it an excellent candidate for the development of both molecular and serological diagnostic tests. The first part of this project was focused on the validation of a real time PCR (qPCR) targeting the lipl32 gene (CDC qPCR), by comparison with two other real time PCR assays (ADDL qPCR and 16S rRNA qPCR) and a gel based PCR assay. Experiments with DNA extracted from 10-fold serial dilutions of Leptospira culture showed that all three qPCR assays were 100-fold more sensitive than the gel based PCR assay. When clinical canine urine samples obtained aseptically were tested, there was complete agreement among the results obtained with all three qPCR. However, when urine samples were collected by free catch method, the 16S rRNA qPCR falsely detected 13.3% of the samples as positive for pathogenic Leptospira spp. Nucleotide sequence analysis of the DNA fragments amplified in the 16S rRNA qPCR indicated that the assay detected a bacterial species that was previously associated with the normal skin surface of animals. The results of this study suggest that the real time PCR assays targeting the lipl32 gene are very specific for detection of pathogenic Leptospira species. The second part of this project involved using the purified recombinant LipL32 as an antigen in ELISA for detection of pathogenic Leptospria specific IgG in serum of animals suspected to have leptospirosis. The gene encoding LipL32 was amplified using a custom-designed primer-pair and cloned in the expression vector, pGEX. The recombinant protein was expressed in fusion with glutathione-S-transferase and purified via affinity chromatography using glutathione magnetic beads. Serum samples from 42 dogs and 100 horses were tested in the ELISA to determine the presence of LipL32-specific IgG. MAT titers of all the samples against 6 serovars were also determined. The results indicated that there was no correlation between the MAT titers and the levels of LipL32-specific IgG in serum samples. Future studies should focus on utilizing a defined set of serum samples to determine the appearance of LipL32-specific IgG in serum following vaccination or exposure to various pathogenic Leptospira species.

Degree

M.S.

Advisors

Vemulapalli, Purdue University.

Subject Area

Microbiology|Immunology

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