# Comparison between ELISA and the Microscopic Agglutination Test for the Diagnosis of Bovine Leptospirosis

I.M. El Jalii<fnref n="1" />

**Keywords**

Cattle – Leptospirosis – Diagnosis – ELISA.

**Summary**

An indirect enzyme-linked immunosorbent assay (ELISA) was compared with the microscopic agglutination test (MAT) for the diagnosis of bovine leptospirosis. A total of 170 random bovine serum samples were collected from cattle farms in Malaysia. The serum samples were examined for the presence of antibodies to leptospires using ELISA and MAT. Ninety-six (56.5%) sera were positive to ELISA, while 47 (27.7%) were positive to MAT. All samples positive to MAT were positive to ELISA, while there was no sample positive to MAT and negative to ELISA. ELISA showed 100% sensitivity compared to MAT. ELISA appears as a better alternative to MAT for the diagnosis of bovine leptospirosis.

## INTRODUCTION

Leptospirosis is a serious zoonotic disease with important veterinary and public health impacts (7). The disease is caused by *Leptospira interrogans*. It causes important economic losses in livestock. Although it is usually mild and often subclinical, it can lead to great losses due to abortion, stillbirth, infertility, mastitis, weak progeny, decreased milk production and, with certain leptospiral serovars, death (4, 13).

Diagnosis of leptospirosis based on serology is the most suitable for the rapid testing of a large number of individuals (5, 7, 11). Serodiagnostic methods used for leptospirosis included the microscopic agglutination test (MAT), hemagglutination (HA) test, fluorescent antibody (FA) and complement fixation test (CFT). Drawbacks of most of these serological tests led to the rapid development of the enzyme-linked immunosorbent assay (ELISA) in the diagnosis of bovine leptospirosis. Different types of ELISA have

been developed and were found to be useful for the diagnosis of leptospirosis (1, 10, 12, 14). The main objective of this study was to compare ELISA and MAT as screening tests for diagnosis of bovine leptospirosis.

## MATERIALS AND METHODS

### Test sera

One hundred and seventy serum samples were collected from two dairy farms in Malaysia: 70 from the University Putra Malaysia Farm and 100 from the Malaysian Agriculture Research and Development Institute (Bahang) Farm. The samples were random and the animals examined were of both sexes with age range from six months to four years. All serum samples were examined for antibodies to *Leptospira* by ELISA and MAT.

### ELISA

The antigen used with ELISA was a genus-specific antigen (lipopolysaccaride) derived from boiling leptospiral culture (serovar *hardjo*). Goat antibovine IgG labeled with horseradish peroxidase was used as conjugate. An indirect ELISA technique was used based on protocols by El Jalii et al. (6). It was carried out with a

<footnote n="1">1. Department of Clinical Studies, Faculty of Veterinary Medicine and Animal Resources, King Faisal University, Postal code 31982, PO box 1757, Al Hasa, Saudi Arabia.
Tel.: +966 35 81 66 00; Fax : +966 35 81 66 35
E-mail: eljalii@yahoo.com</footnote>
working volume of 100 µl of each reagent. The 96 wells of micro-
titration plates (Dynex, Immulon, USA) were coated with 100 µl of
1/1600 *hardjo* LPS antigen in 0.05 M carbonate/bicarbonate buffer
pH 9.6, incubated at 4°C overnight, and washed four times with
washing buffer containing 0.05% (v/v) Tween 20 in PBS pH 7.0
using a microplate washer (Nunk Immuno Washer 8, Denmark).
Then, 100 µl of 2% BSA fraction V (Sigma, UK) were added to
the wells to prevent non-specific binding and incubated for 2 h at
37°C. After washing four times, 100 µl of known bovine positive
and negative sera (1/200 diluted sera in PBS pH 7.2 containing
0.03% Tween 20) as controls and the test sera were added to wells
in duplicate and incubated at 37°C for one hour. The plate was
then washed four times and 100 µl of pre-diluted goat anti-bovine
peroxide conjugated immunoglobulin (IgG) (KPL, USA) in PBS-
Tween 20 were added and incubated at 37°C for one hour, washed
four times, and 100 µl of 2,2-azino-bis(3-ethylbenzthiazoline-6-sul-
fonic acid) and 2 mM hydrogen peroxide in 0.05 M citrate buffer
pH 4.0 substrate were added to each well and incubated at room
temperature in the dark for 10 min. The results were read with the
aid of a spectrophotometer (Titretek Multiskan Plus, Finland) using
405 nanometers wavelength. Sera were reported as positive if their
optical density (OD) readings were higher than the OD readings of
the mean negative controls plus three standard deviations.

MAT

The antigens used with MAT were made up of 4-8 day-old live cul-
tures of the following serovars: *hardjo*, *canicola*, *icterohemorrhagiae*,
*australis*, *pomona* and *patoc* strain *patoc1*. MAT was performed as
described by Cole et al. (5) with some modifications. Each serum
sample was initially diluted (1/25) with phosphate buffer saline
(PBS) pH 7.2 in a microtitration plate (Greiner Labrotechnik), 25 µl
of PBS were placed into each well of the plate and an equal volume
of the diluted serum sample was placed in the first row (A row) of
the plate. The diluted serum which was then 1/50 was serially diluted
(two-fold) from A row to H row using a hand-held 0.025 ml micro-
diluter. Then, 25 µl of the live antigens (4-8 day-old cultures con-
taining 10⁸ leptospires/ml) were added to each well. Thus, each well
contained an equal volume of the diluted serum sample and the anti-
gen. For each serum sample tested, there were eight dilutions ranging
from 1/100 to 1/12,800. The plate was gently shaken for 15-20 s to
mix the contents, covered to exclude debris and prevent evaporation,
and incubated for one hour at 37°C. The test was read by transfer-
ring a drop from each well onto a glass microscope slide. The drops
were examined by dark-field microscopy (x200). A positive reaction
was regarded as one in which 50% or more of the antigen (live lepto-
spires) were agglutinated. The titer end point was taken as the last
well in which 50% or more agglutination was observed.

Statistical analysis

Stata 98/NT was used for analysis.

■ RESULTS

Out of the 170 serum samples examined by MAT, 47 (27.7%) were positive to MAT, while 123 (72.3%) were negative. Forty (85.1%) of MAT positive animals were found to be infected by serovar *hardjo*, which is a predominant infecting serovar. Five (10.6%) were infected by *pomona*, and two (4.3%) by *australis*.

Out of the 170 serum samples examined by ELISA, 96 (56.5%) were positive, while 74 (43.5%) were negative.

When comparing MAT and ELISA, 96 (56.5%) were positive to
ELISA, while 47 (56.5%) were positive to MAT.

All samples which were positive to MAT were positive to ELISA.

There was no sample positive to MAT and negative to ELISA. For-
ty-nine (28.8%) were positive to ELISA and negative to MAT.

■ DISCUSSION

Leptospirosis is one of the diseases with economic impact, so its
diagnosis and serosurveillance are very important for any con-
trol program. Many tests have been used in the field as screening
tests of leptospirosis. The present study was designed to compare
ELISA and MAT for screening of bovine leptospirosis. Serological
evidence of three leptospiral serovars was revealed from animal
sera tested, i.e. *hardjo*, *pomona* and *australis*, with serovar *hardjo*
as the most prevalent (85.1% prevalence). In Malaysia, 38 sero-
vars from 13 serogroups have been known to occur (2). The high
prevalence of *Leptospira serovar hardjo* infection in this study was
significant because it supported previous studies on animal lept-
ospirosis in Malaysia which reported that cattle maintained *Lept-
ospira serovar hardjo* (3, 8).

Using MAT, three leptospiral antibodies were revealed in this
study. ELISA detected positive samples to leptospirosis from
sera infected with these three serovars. The antigen used in this
study was heat-extracted and was prepared from a single serovar
(*hardjo*). It permitted the detection of infection caused by differ-
ent serovars. The use of a single serovar to prepare the antigen for
ELISA appeared reliable and easy. This antigen was stable and
could be stored for a long time either in liquid state or coated onto
polystyrene plates. On the other hand, MAT used live leptospires
belonging to different serovars, which had to be propagated and
subcultured continuously to carry out the test. This was very tedi-
ous and created a risk of infection to laboratory personnel. ELISA
showed 100% sensitivity compared to MAT. No sample positive
to MAT was negative to ELISA. This indicated that ELISA was
more sensitive than MAT and offered many advantages over MAT.
The technique is semi-automated, easy and reproducible. ELISA
can potentially be used as a screening test for leptospirosis (9, 15).
In conclusion, ELISA was sensitive and could detect antibodies
to multiple pathogenic *Leptospira* serovars. It is a good assay for
bovine leptospirosis screening.

Acknowledgments

I am grateful to Prof. A.R. Bahman from the Faculty of Veterinary Medicine, University Putra, Malaysia, for advice and help in sample collection and laboratory facilities.

REFERENCES

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2. BAHAMAN A.R., 1988. A serological and bacteriological study of leptospiral infection in domestic animals in Peninsular Malaysia. PhD Thesis, University Putra, Selangor, Malaysia.

3. BAHAMAN A.R., IBRAHIM A.L., 1987. A short review of animal leptospirosis with special reference to Malaysia. *Trop. Biomed.*, **4**: 93-99.

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Reçu le 02.04.2007, accepté le 15.09.2008

Résumé

El Jalii I.M. Comparaison entre le test Elisa et le test de microagglutination dans le diagnostic de la leptospirose bovine

Un test Elisa indirect a été comparé au test de microaglutina-
nation (MAT) dans le diagnostic de la leptospirose bovine. Au
total 170 échantillons sériques aléatoires on été prélevés sur
des bovins dans des fermes en Malaisie. L'examen des échan-
tillons a porté sur la recherche d'anticorps contre *Leptospira* en
utilisant Elisa et MAT. Quatre-vingt-seize (56,5 p. 100) échan-
tillons ont été positifs à l'Elisa, alors que 47 (27,7 p. 100) l'ont
été au MAT. Tous les échantillons qui ont été positifs au MAT
l'ont aussi été à l'Elisa ; il n'y a pas eu d'échantillon positif au
MAT et négatif à l'Elisa. La sensibilité d'Elisa a été de 100 p.
100 comparée à celle de MAT. Elisa apparaît comme étant la
meilleure technique des deux dans le diagnostic de la leptos-
pirose bovine.

Mots-clés : Bovin – Leptospirose – Diagnostic – Test Elisa.

Resumen

El Jalii I.M. Comparación entre el ensayo de inmunoabsor-
bente ligado a enzimas y el test de microaglutinación para el
diagnóstico de la leptospirosis bovina

Se comparó un ensayo inmunoabsorbente ligado a enzimas (ELISA) indirecto con el test de microaglutinación (MAT) para el diagnóstico de la leptospirosis bovina. Se colectó al azar un total de 170 sueros bovinos proveniente de ganado de fincas en Malasia. Las muestras de suero se examinaron para la presencia de anticuerpos de leptospirosis usando ELISA y MAT. Noventa y seis (56,5%) de los sueros fueron positivos con ELISA, mientras que 47 (27,7%) fueron positivos con MAT. Todas las muestras positivas mediante MAT fueron positivas mediante ELISA, mientras que ninguna muestra fue positiva mediante MAT y negativa mediante ELISA. ELISA mostró 100% de sensitividad comparado con el MAT. ELISA parece ser una mejor alternativa que MAT para el diagnóstico de la leptospirosis bovina.

Palabras clave: Ganado bovino – Leptospirosis – Diagnóstico – ELISA.