research and diagnosis of leptospirosis. Julie Collins-Emerson*, - PowerPoint PPT Presentation

Utility of diagnostic tests for research and diagnosis of leptospirosis. Julie Collins-Emerson*, Fang Fang, Jackie Benschop, Cord Heuer & Peter Wilson. OIE Collaborating Centre for Veterinary Epidemiology and Public Health

Key questions: 1. What tests are most successful for detecting Leptospira in: - different specimen types - different species (sheep/cattle) - at different stages of the disease

Key questions: 1. What tests are most successful for detecting Leptospira in: - different specimen types - different species (sheep/cattle) - at different stages of the disease 2. How well do the same diagnostic tests run under the “ideal” conditions attainable in a research laboratory compare to those performed within the “commercial reality” of a veterinary diagnostic laboratory?

Key questions: 1. What tests are most successful for detecting Leptospira in: - different specimen types - different species (sheep/cattle) - at different stages of the disease 2. How well do the same diagnostic tests run under the “ideal” conditions attainable in a research laboratory compare to those performed within the “commercial reality” of a veterinary diagnostic laboratory? 3. What are the sources of variation with diagnostic test results?

Diagnostic tests utilised - Microscopic agglutination test (MAT) by Becca Chandler - Dark field microscopy (DFM) - Real-Time PCR (RT-PCR) (two chemistry types – SYTO9 vs. TaqMan probe) vs. - culture

Sources of possible variation examined. • host species • serovar strain • stage of disease • diagnostic sample type • kind of diagnostic test • sample preparation e.g. DNA extraction • technical operator both within and between labs

Strategy Two main lines of enquiry: 1) Appraise test results: different diagnostic samples • • at different stages of disease in different host species • ……under controlled experimental conditions

Strategy Two main lines of enquiry: 1) Test result comparisons: diff. diagnostic samples • • at diff. stages of disease in diff. host species • ……under controlled experimental conditions 2) Inter-lab comparisons of diagnostic tests i.e. research (HLRL) vs. commercial (GV) results on the same samples

4 Controlled trials Note: Involvement in the 4 trials (A,B,C &D) was opportunistic • As we “piggy - backed” on commercial trials, there was • little opportunity to influence the design of the trials. Accepted their design was not ideal but did allow access • to animals under controlled challenge conditions All trials had animal ethics approval (Kaiawhina Animal Ethics Committee, Palmerston North, New Zealand)

The trials • Trial A used to passage and “hot up” suitable lab - stored field isolates for challenge trials i.e. selection for virulence. • L. borpetersenii sv. Hardjobovis (H.bovis) and L. interrogans sv. Pomona (Pom) used • Trials to serve as pilots for later vaccination trials

Trial A – initial passage trial (pilot) Cultures used: H.bovis a = isolate from commercial vet. path. lab., (GV), origin unknown H.bovis b = isolated from a deer by Hopkirk Leptospirosis Research Laboratory (HLRL) 2 x H.bovis strains Pom a = isolate from commercial vet. path. Lab. (GV), origin unknown Pom b = isolated from a sheep by Hopkirk Leptospirosis Research Laboratory (HLRL) 2 x Pom strains

Trial A – initial passage trial (Pilot) Challenged on 3 success days Pen 1 Pen 2 A1 A6 Pom a H.bovis a A7 A3 Pen 3 Pen 4 A5 A2 ‡ Pom b H.bovis b A8 A4

Trial A – initial passage trial (Pilot) Samples taken: • Urine - (for culture, PCR, DFM & MAT) • Kidney - (for culture, PCR & DFM) • blood - (for serology) • Length of trial = 42 days

Trial A – Results Detection of leptospires in urine on each sampling day after challenge, and kidney on Day 42, by culture (C), PCR (P) and Dark Field Microscopy (D) and antibody titres against serovars Hardjobovis and Pomona by MAT for sheep Trial A. Urine* Kidney MAT titre Sheep Serovar Day Day Day ID inoculated 19 † 19 22 27 30 33 42 0 1 2 3 42 Hardjobovis a − − − − − − − − − − − A1 D ‡ Hardjobovis b − − − − − A2 P P,C C P P,C,D 1:100 1:192 Hardjobovis a − − − − − − − − − − − − A3 Hardjobovis b − − − − − A4 C C C C P,C 1:1600 1:768 Pomona b − − − − − − − − − A5 P,D 1:24 1:24 Pomona a − − − − − − − − − − − − A6 − − − − − − − − − − − Pomona a A7 D − − − − − − − − − − − − Pomona b A8 * N.B. Urine not collected on Day 0 ; † MAT performed by GV rather than HLRL ‡ This passage isolate was used for challenge in Trial B

Trial A – Results • HLRL H.bovis a most successful for PCR, DFM. Also only isolate to be successfully cultured after passage. • HLRL marginally better results for PCR and DFM detection than GV cultures • Neither Pom strains successfully passaged

Trial A conclusions • Either challenge method not optimal for Pom strains, or, not particularly virulent. • H.bovis b & Pom b from HLRL more successful – selected for further trialing. • Use passaged culture from sheep A2 for following H.bovis trials.

Trial B results B: H.bovis isolated from sheep ‡ A2 into 8 x Detection of Leptospira in urine and kidney and MAT results Urine Kidney MAT titre against serovar Hardjobovis Sheep ID Day Day Day 0 5 8 12 14 21 28 37 42/15 a 0 5 8 12 14 21 28 37 B1 − − − − − P − − − 1:384 1:192 1:384 1:48 1:96 B2 − − − − − − − − D − − 1:96 1:1536 1:768 1:384 1:192 1:48 B3 − − − − − − D D − − 1:48 1:1536 1:768 1:96 1:48 1:24 B4 a − − − − − − − − 1:96 1:768 1:768 B5 − − − − − − − D D − − 1:48 1:3072 1:1536 1:384 1:192 1:192 B6 P,C P,C P,C P,C P,C,D P,C,D P P,C,D P,D 1:24 1:48 1:192 1:96 − 1:192 1:96 1:24 B7 − − − − − D − − D − − 1:384 1:768 1:1536 1:384 1:384 1:96 B8 − − − − − − − D D − − 1:768 1:1536 1:768 1:192 1:96 1:96 N.B. B4 a died on Day15 at which time the kidney was sampled. C = culture, P = PCR, D = dark field microscopy, - = neg. result

Trial B results • Not successful in establishing an infection. • No Lepto detected in any blood samples • 1 PCR pos. kidney • All animals “saw” the antigen • *Animal B6 – detected Lepto in urine and kidney each sampling by PCR and culture – silent carriage! • Therefore techniques successful

Trial B conclusions • The MAT results suggested all animals “saw” the antigen • However, not particularly successful at establishing infection • *Animal B6 – detected Lepto in urine and kidney at each sampling by PCR and culture – silent carriage! Established the PCR and culture as consistent and reliable in • this case.

Trial C results Pom human NZ isolate into 16 x Blood Serum Urine Kidney MAT a titre against serovar Pomona Inoculated Sheep Day Day Day Day Day ID strains 0 3 5 7 14 b 0 3 5 7 14 7 14 21 28 35 42 42 0 5 7 14 28 42 − − − − − − − − − − − − − C1 P P P P,C C P 1:96 1:3072 1:800 1:50 − − P − − − − − − − − − − − − C2 P P P P 1:96 1:192 1:50 1:25 C3 − − − − − − − − − − − − − − D P C P,D D 1:24 1:768 1:100 1:50 − C − − − − − − − − − C4 P P P,C P,C P,C,D P,C,D P,C P,C,D 1:48 1:3072 1:800 1:25 LPC − C − − − − − − − − C5 P P P P,C P,C,D P,C P,C,D P,C P,C 1:24 1:1536 1:800 1:50 04/08 C6 − C − − − − − − − − − C P P P C C P,C P,C P,C 1:1536 1:800 1:25 − C7 − − − − − − − − P,C P P D P,C P,C P,C,D P,C,D P,C P,C 1:48 1:3072 1:800 1:100 − C − − − − − − − − C8 P P P C P,C P,C P,C P,C P,C 1:48 1:3072 1:200 1:100 − P,C − − − − − − − − C9 P P P C C P,C P,C P,C P,C 1:24 1:384 1:400 1:50 C10 − C − − − − − − − − C P P P P,C P,C P,C,D P,C P,C P,C 1:384 1:400 1:25 C11 − C − − − − − − − − − P P P P,C,D P,C P,C P,C P,C 1:48 1:1536 1:400 1:25 C12 − P,C − − − − − − − − − P C P,C P,C,D P,C P,C P,C 1:24 1:768 1:800 1:100 1:50 LPC 04/04 C13 − − − − − − − − − P,C P P P P,C P,C P,C P,C,D P,C P 1:48 1:1536 1:400 1:100 C14 − − − − − − − − − − P P D C P,C P,C,D C C 1:48 1:768 1:1600 1:100 1:100 C15 − C − − − − − − − − − − P P C P,C P,C P,C P,C P,C,D 1:384 1:800 1:50 C16 − − − − − − − − − − − − − − − − C C C 1:48 1:768 1:400 1:25 a = MAT performed by NZVP Days 14-42, b = blood culture not performed on Day 14

Trial C conclusions recap. • Successful challenge with the human Pom isolates • Lepto first detected by both PCR and DFM on Day 7 and still on Day 42, culture by Day 14 • RT-PCR in serum slightly more successful than in whole blood in this study (but no.s. small). (Covered in detail by Jackie in the previous talk.) • MAT peaking ~ Day 7-Day 14