Veterinary Epidemiology and Zoonosis

Dark field mircoscopic examination and Microscopic agglutination test

EXERCISE-10: DIAGNOSIS OF LEPTOSPIROSIS

• Leptospires are spiral-shaped bacteria, with open hooked ends. They are motile, aerobic and culturable and they measure some 6 to 20 microns long by 0.1 microns in diameter.
• They can be seen under a dark field microscope and pass through filters that block other bacteria.
• Two species are recognized: Leptospira interrogans and L.biflexa. L.interrogans is pathogenic for man and animals.
• L.biflexa is a free-living saprophyte found in surface waters.
• The species of interest as a zoonotic agent is L.interrogans.
• It has more than 200 serologic variants or serovars. Serovars are grouped for convenience into 23 serogroups.
• Human infection is endemic in certain parts of Tamilnadu.
• Farmers, veterinarians, abattoir workers, sewer workers, fish farmers, canal workers, sugar cane cutters and rice field workers are under high risk.
• It causes icteric and non-icteric leptospirosis in human.
• Isolation and identification by culture and observing under Dark field microscopy (DFM).
• Examination of serum and/or urine under DFM.
• Microscopic agglutination test (MAT), a referral test. MAT is a “Gold standard test”.
• Silver impregnation staining and examination of culture media under light microscope.

Figure: Dark field microscope

Diagnosis of leptosirosis by dark field mircoscope

• It is a qualitative test for initial screening of suspected cases of leptospirosis.
• Serum is the choice of material for examination under DFM.
• Test serum should be examined immediately or 3 to 4 hours after the collection.
• Serum should not be refrigerated or should not be transported on ice, because ice-cold condition will affect the motility of the leptospires.

Materials required

Materials required	Quantity and purpose
Test serum (should not be refrigerated or should not be transported on ice)	0.5mL
Micropipettes and tips	For dispensing test serum
Glass slides and cover glass	For microscopic examination
Immersion oil	For viewing under wet DFM and for observing under 100X
Antiseptic solution	For discarding of slides after examination

Procedure

• Place 10µL of suspected serum on a clean glass slide and put cover glass.
• Put a drop of immersion oil on the dark field condenser (if it is wet dark field condenser) and keep the slide over the condenser.
• Examine under DFM at 40X. It needs experience to identify the presence of leptospires.

Interpretation of result

• Leptospires are seen as spiral-shaped with hooked ends against dark background, measure about 6 to 20 microns long by 0.1 microns in diameter. They are motile with screwing movement and often form different shapes (secondary structures) like ‘S’ or ‘V’ or ‘U’ or ‘L’ or ‘C’ or ‘hair-pin shaped’.

Precaution

• Care must be taken to avoid touching of test serum. After examination slide must be placed in to the antiseptic solution or it should be autoclaved and then discarded.

Diagnosis of leptosirosis by mircoscopic agglutination test

• Microscopic agglutination test (MAT), a referral test. MAT is a “Gold standard test” as per World Organization for Animal Health (Office International des Epizooties).
• It is a quantitative test.
• Test serum can be stored at refrigeration temperature.
• MAT requires live leptospira liquid culture.
• MAT can be performed as serovar-specific Leptospire or with pooled Leptospira serovars prevalent in that area.
• A four to eight day old liquid culture of live Leptospires incubated at room temperature containing 2x10⁸ Leptospires per mL without clumps is used as antigen for MAT

Materials required

Materials required	Quantity and purpose
Test serum	0.5mL
Live leptospira culture (either pooled antigen or individual culture medium)	0.5mL
Phosphate buffer saline	5mL
‘U’ bottom 96-wells microtitre plate	For making two-fold dilution of serum and incubation with live leptospira culture
Micropipettes and tips	For dispensing test serum and leptospira culture medium
Glass slides	For microscopic examination
Biological incubator	To incubate serum-leptospira mixture
Immersion oil	For viewing under wet DFM
Antiseptic solution	For discarding of slides after examination

Procedure

• Add 96µL of sterile phosphate buffer saline (pH 7.2) in the 1^st well and 50µL in the next four wells in a 96-wells microtitre plate.
• Dilute 4µL of test serum in the 1^st well (1:25) and mix thoroughly. Transfer 50µL from the 1^st well to the 2^nd well and mix and continue up to 5^th well. Discard 50µL from the 5^th well. The dilution rate is 1:25, 1:50, 1:100, 1:200 and 1:400.
• Add constant amount of 50µL of antigen pool (To know the serovar-specific MAT titre individual leptospira culture should be added separately). The final dilution after the addition of antigen pool is 1:50 to 1:800.
• Positive and negative controls should be included.
• Incubate the contents of the microtitre plate at room temperature (37°C) for 3 hours.
• At the end of the incubation, take a loop full of serum-antigen mixture on a clean glass slide beginning from highest dilution and examine under DFM without coverglass at 40X.

Figure: Microscopic agglutination test showing Leptospire-specific agglutinated clumps

Interpretation of result

• The end point of an agglutination reaction is taken as the highest dilution at which 50 per cent of the Leptospires had agglutinated. The reciprocal of end point formed the titre.
• Titre of 1:100 is considered as positive for leptospirosis.

Precaution

• Care must be taken to avoid touching of test serum and Leptospira live culture. After examination slide and microtitre plate must be placed in to the antiseptic solution or it should be autoclaved and then discarded.