The Microagglutination Test (MAT), the reference serological test, was used for processing samples, considering a titer of ≥1:200 as a criterion of positivity. This is the cut-off point used in cattle in Argentina since leptospirosis is an endemic disease and because cattle are vaccinated in areas where this agent is present (17 ). Antibodies against pathogenic Leptospira were detected by the MAT in the Leptospirosis Laboratory, Department of Rural Zoonosis (Ministry of Health of Buenos Aires Province), according to WOAH (18 ) protocols.
A panel of live antigens of ten Leptospira spp. reference strains were used: L. interrogans serogroup Canicola serovar Canicola strain H. Utrecht IV, serogroup Hebdomadis serovar Hebdomadis strain Hebdomadis, serogroup Icterohaemorrhagiae serovar Copenhageni strain M20, serogroup Pomona serovar Pomona strain Pomona, serogroup Pyrogenes serovar Pyrogenes strain Salinem, serogroup Sejroe serovar Wolfii strain 3,705 and serogroup Sejroe serovar Hardjo strain Hardjoprajitno, L. borgpeterseni serogroup Ballum serovar Castellonis strain Castellon 3 and serogroup Tarassovi serovar Tarassovi strain Perepelitsin and L. kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain Castellon 3. This panel was developed at 28–30°C in the Ellinghausen-McCullough-Johnson-Harris (EMJH) medium with no more than 15 days of growth. Serial serum dilutions were performed with phosphate-buffered saline (PBS, pH 7.2) starting from 1:100 dilution. The plates were incubated at 37°C for 90 min. After incubation, the serum-antigen mixtures were checked for agglutination under a dark field microscope. Tests were interpreted as positive when agglutination at ≥ 1:200 of at least 50% of the leptospires for any serogroup was observed. The highest serum dilution with >50% agglutination or ≤50% free leptospires, compared to the negative control, was considered the endpoint titer of quantitative MAT.
A panel of live antigens of ten Leptospira spp. reference strains were used: L. interrogans serogroup Canicola serovar Canicola strain H. Utrecht IV, serogroup Hebdomadis serovar Hebdomadis strain Hebdomadis, serogroup Icterohaemorrhagiae serovar Copenhageni strain M20, serogroup Pomona serovar Pomona strain Pomona, serogroup Pyrogenes serovar Pyrogenes strain Salinem, serogroup Sejroe serovar Wolfii strain 3,705 and serogroup Sejroe serovar Hardjo strain Hardjoprajitno, L. borgpeterseni serogroup Ballum serovar Castellonis strain Castellon 3 and serogroup Tarassovi serovar Tarassovi strain Perepelitsin and L. kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain Castellon 3. This panel was developed at 28–30°C in the Ellinghausen-McCullough-Johnson-Harris (EMJH) medium with no more than 15 days of growth. Serial serum dilutions were performed with phosphate-buffered saline (PBS, pH 7.2) starting from 1:100 dilution. The plates were incubated at 37°C for 90 min. After incubation, the serum-antigen mixtures were checked for agglutination under a dark field microscope. Tests were interpreted as positive when agglutination at ≥ 1:200 of at least 50% of the leptospires for any serogroup was observed. The highest serum dilution with >50% agglutination or ≤50% free leptospires, compared to the negative control, was considered the endpoint titer of quantitative MAT.
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