Laboratory Diagnosis and TreatmentLaboratory Diagnosis and Treatment

LeptospirosisLeptospirosis in Livestockin LivestockDuangjai Suwancharoen

Leptospirosis CenterLeptospirosis CenterNational Institute of Animal Health

Department of Live Stocks Development

Leptospirosis is an economically important p p y pzoonotic bacterial infection of livestock th tthat causes:

- abortions- stillbirths

infertility- infertility- loss of milk production.

In particular regions, different leptospiral sero ars are pre alent and are associatedserovars are prevalent and are associated with one or more maintenance hosts.

Diagnosis of leptospirosis is dependant on: - a good clinical - vaccination history- vaccination history- the availability of diagnostic

testing at a laboratory with experience in thediagnosis of leptospirosis.diagnosis of leptospirosis.

Coordination between the diagnostic l b d h i i i i dlaboratory and the veterinarian is required to maximize the chances of making an accurate diagnosis.It is advisable to contact the diagnosticIt is advisable to contact the diagnostic laboratory prior to submission of samples to assure that appropriate samples are collected and that the samples arrive at the diagnostic glaboratory in suitable condition

Diagnostic tests for leptospirosis can be separated into those designed:

- detect antibodies against the organism - detect the organism or its’ DNA in tissues or body fluids of animalsor body fluids of animals.

E h f th di ti d- Each of the diagnostic procedures, has a number of advantages and gdisadvantages.

S f h ff f l k- Some of the assays suffer from a lack of sensitivity and others are prone toof sensitivity and others are prone to specificity problems

-Therefore, no single technique can be d d f i h li i l it tirecommended for use in each clinical situation.

Use of a combination of tests allows maximum-Use of a combination of tests allows maximum sensitivity and specificity in establishing the diagnosis.

Serological testing is recommended in each- Serological testing is recommended in each case, combined with one or more techniques to id if h i i i b d fl ididentify the organism in tissue or body fluids.

Specimens

Urine Kid SerumUrine Kidney(and other body

tissues)

Tissue Sections

Serum

Serological TestsA i l I l tiC t if d Tissue SectionsFluorescent antibody test

(FAT)

Serological Tests

- Macroscopic

agglutination

Animal InoculationCentrifuged deposit

Gerbils(screening)

- Microscopic

agglutination

Tissue suspensionsDirect Microscopy

- Dark - field

microscopy

Gerbils

Weanling hamsters

Young guinea-pigs

test (MAT)

- ELISA

- Complement

- Fluorescent

antibody technique

(FAT)

Blood at febrile stage

Complement

fixation test(FAT)

Culture 30oC Direct Microscopy

- Dark-field Identification

MAT

( i ifi i )

microscopy

- Fluorescent

tib d t h i (FAT)(using known specific antisera) For serogroup and then serovar

antibody technique (FAT)

Summary of the laboratory procedures for the diagnosis of leptospirosis.

Specimens

Urine Kid SerumUrine Kidney(and other body

tissues)

Tissue Sections

Serum

Serological TestsA i l I l tiC t if d Tissue SectionsSilver impregnation Immunoperoxidase

Serological Tests

- Macroscopic

agglutination

Animal InoculationCentrifuged deposit

Gerbils(screening)

- Microscopic

agglutination

Tissue suspensionsDirect Microscopy

- Dark - field

microscopy

Gerbils

Weanling hamsters

Young guinea-pigs

test (MAT)

- ELISA

- Complement

- Fluorescent

antibody technique

(FAT)

Blood at febrile stage

Complement

fixation test(FAT)

Culture 30oC Direct Microscopy

- Dark-field Identification

MAT

( i ifi i )

microscopy

- Fluorescent

tib d t h i (FAT)(using known specific antisera) For serogroup and then serovar

antibody technique (FAT)

Summary of the laboratory procedures for the diagnosis of leptospirosis.

Serologic testsSerologic tests- The microscopic agglutination test p gg(MAT) is the most commonly used technique for diagnosing leptospirosistechnique for diagnosing leptospirosis in animals. Serology is inexpensive, reasonably sensitive, and widely available.available.

A ti P l Antigen Panel1. Australis2 Autumnalis

9. Grippotyphosa10 Hebdomadis

17. Pomona18 Pyrogenes2. Autumnalis

3. Ballum10. Hebdomadis11. Icterohaemorrhagiae

18. Pyrogenes19. Ranarum

4. Bataviae5 C i l

12. Javanica13 L i i

20. Sarmin21 S j5. Canicola

6. Celledoni13. Louisiana14. Manhao

21. Sejroe22. Shermani

7. Cynopteri 15. Mini 23. Tarassovi8. Djasiman 16. Panama 24. Patoc

ิ ิ ี่ ิ ิ ี่แอนติเจนชนดิที 1-12 แอนติเจนชนดิที 13-24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24C1

C1C2

C1C2

2S 1

2

234

S

345

56

56 C1 = positive control

C2 = negative controlC egat e co t oS = serum samples

MAT developmentMAT development

- Reading result: on slide to microplatePeriod of time: 2 da s to 1 da to 3 hrs- Period of time: 2 days to 1 day to 3 hrs

- Monitoring microscope: using program computer reading

Test kits:

ELISA f ifi- ELISA for serovar specific: Leptospira Hadjo ELISA in Cattlep p jLeptospira Bratislava cELISA in Pig

Test kits:Test kits:

- Bovine Leptostick (Hardjo-specific) in milk milk

Interpretation of Leptostick Resultsp f p

Immunostick A: Negative resultImmunostick B: Positive ResultImmunostick B: Positive Result

A BA B

In cases of acute leptospirosis, a fourfold rise in antibody titer is often observed inrise in antibody titer is often observed in paired serum samples. However, cattle are commonly actively infected and shedding serovar Hardjo with antibody titers < 100.j y

Leptospiral antibody titers are often steady or decreasing at the time of abortion andor decreasing at the time of abortion and up to 50% of cows aborting due to serovar Hardjo will be seronegative at the time of abortionabortion. Therefore, a low antibody titer does not , ynecessarily rule-out a diagnosis of leptospirosisleptospirosis.

Specimens

Urine Kid SerumUrine Kidney(and other body

tissues)

Tissue Sections

Serum

Serological TestsA i l I l tiC t if d Tissue SectionsFluorescent antibody test

(FAT)

Serological Tests

- Macroscopic

agglutination

Animal InoculationCentrifuged deposit

Gerbils(screening)

- Microscopic

agglutination

Tissue suspensionsDirect Microscopy

- Dark - field

microscopy

Gerbils

Weanling hamsters

Young guinea-pigs

test (MAT)

- ELISA

- Complement

- Fluorescent

antibody technique

(FAT)

Blood at febrile stage

Complement

fixation test(FAT)

Culture 30oC Direct Microscopy

- Dark-field Identification

MAT

( i ifi i )

microscopy

- Fluorescent

tib d t h i (FAT)(using known specific antisera) For serogroup and then serovar

antibody technique (FAT)

Summary of the laboratory procedures for the diagnosis of leptospirosis.

Localisation in the kidneyLocalisation in the kidneyLocalisation in the kidneyLocalisation in the kidney

W. Ellis

Tissue SectionsFluorescent antibody test

(FAT)

Leptospirosis CenterNational Institute of Animal Health

SAMPLES

Serum

Antibodies

Urine, Organs, Water, Soil,Blood, Semen

Antibodies detection

MAT ( Microscopic Agglutination Test)

Antigen detection Nucleic acid detection

Isolation

Pure Leptospires

-PCR ( Polomerase Chain Reaction) -LAMP ( Loop mediated isothermal Amplification)

-Realtime PCR

Identification

Realtime PCR

Pure DNA Leptospires

-CAAT (Cross Absorption Agglutination Test)

-PFGE (Pulsed - Field Gel Electrophoresis)-Sequencing

Identification

Sequencing

IsolationIsolationIsolationIsolation

Develop medium preparation by using various Develop medium preparation by using various ibi i ibi i antibiotic antibiotic

Urine and Organs used Urine and Organs used 4 4 kinds: Mkinds: M11, M, M22, M, M33, M, M44gg(OIE (OIE LeptospirosisLeptospirosis Reference Reference Laboratory,VeterinaryLaboratory,Veterinary SciencesSciences DivisionDivision, , Belfast, Belfast, NorthernNorthern IrelandIreland))

Environment used: STAFF Environment used: STAFF ((sulfamethoxazolesulfamethoxazole,,trimethoprim, amphotericin B, trimethoprim, amphotericin B, fosfomycinfosfomycin, , 55--

FU) FU) FU) FU) ((ChakrabortyChakraborty A, A, et alet al., ., A novel combination of selective agents for isolation of A novel combination of selective agents for isolation of LeptospiraLeptospira species,species, MicrobiolMicrobioland and ImmunolImmunol 20112011; ; 5555((77): ): 494494––501501.).)

ImmunochromatographyImmunochromatography(IC)(IC)ImmunochromatographyImmunochromatography(IC)(IC)

Detection of Detection of leptospiresleptospires: on Processing: on Processing

Leptospirosis CenterNational Institute of Animal Health

SAMPLES

Serum

Antibodies

Urine, Organs, Water, Soil,Blood, Semen

Antibodies detection

MAT ( Microscopic Agglutination Test)

Antigen detection Nucleic acid detection

Isolation

Pure Leptospires

-PCR ( Polomerase Chain Reaction) -LAMP ( Loop mediated isothermal Amplification)

-Realtime PCR

Identification

Realtime PCR

Pure DNA Leptospires

-CAAT (Cross Absorption Agglutination Test)

-PFGE (Pulsed - Field Gel Electrophoresis)-Sequencing

Identification

SequencingMLST

- Polymerase chain reaction (PCR) tests can b d t d t t l t i l DNA i li i lbe used to detect leptospiral DNA in clinical samples.

I l PCR i f i i li bl- In general, PCR testing of urine is more reliable than testing of tissues. PCR assays are able to detect the presence of leptospires but are not able to determine the infecting serovar. - PCR can be a sensitive and specific technique for the diagnosis of leptospirosis. g p p- Unfortunately, the process is exquisitely sensitive to contamination with exogenous leptospiral DNAto contamination with exogenous leptospiral DNA and, therefore, may be prone to false-positive reactionsreactions.

LAMP ( Loop mediated isothermal Amplification )

TreatmentTreatment

TreatmentTreatmentTreatmentTreatment

Effect of streptomycin treatment on the shedding of and the serologic t L pt pi i t h dj bt h dj b i iresponses to Leptospira interrogans serovar hardjo subtype hardjobovis in

experimentally infected cows

k d l h kMarijke J. Gerritsen, Metje J. Koopmans and Tom OlyhoekDepartment of Bacteriology, Central Veterinary Institute, Lelystad, Netherlands

Evaluation of antibiotics for treatment of cattle infected with Leptospira borgpetersenii serovar hardjoborgpetersenii serovar hardjo

David P. Alt, DVM, PhD; Richard L. Zuerner, PhD; Carole A. Bolin, DVM, PhDPhD

TreatmentTreatment

A single injection of

Dihydrostreptomycin-penicillin G (25 mg/kg [11.4 mg/lb], IM))

Oxytetracycline (20 mg/kg [9 mg/lb] of body weight, IM), Tilmicosin (10 mg/kg [4.5 mg/lb], SC)

Multiple injections of Amoxycillin (15 mg/kg IM) two injection (48 hrs apart)Amoxycillin (15 mg/kg, IM), two injection (48 hrs apart).Ceftiofur sodium (2.2 or 5 mg/kg [1 or 2.3 mg/lb], IM,once daily for 5 days, or 20 mg/kg, IM, once daily for 3 y y , g g, , y

days).

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