This was calculated according to the equation below. The 2nd criterion used to determine acceptability of the results was, that for any plate, the mean % HPC value PB-22 of only 1 1 of the controls PB-22 was allowed to fall outside of the upper and lower limits (mean % HPC of at least 25 tests 2s, respectively, for each control). 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovarhardjobovisas early as 1 week postinoculation == Rsum == Une preuve ELISA indirecte a t dveloppe afin de dtecter chez les bovins des anticorps envers des srovars pathognes de Leptospira, tels canicola, copenhageni (reprsentant icterohaemorragiae), grippotyphosa, hardjobovis, pomona et serjoe. Lantigne utilis dans cette preuve tait un mlange soniqu de quantits gales de cellules entires tues de chacun des 6 srotypes mentionns. Un anticorps monoclonal de souris dirig contre les immunoglobulines bovines de type IgG1 et conjug la peroxydase de raifort a t utilis pour la dtection des anticorps lis. Cette preuve a t value avec des srums (n =3107) qui donnaient un rsultat ngatif lors de lpreuve dagglutination microscopique (MAT) ( une dilution de 1:100) pour chacun des 6 srovars mentionns prcdemment et des srums (n =601) qui taient MAT-positifs ( une dilution de 1:100) pour 1, ou toute combinaison des 6 srovars lists. De plus, des chantillons de srum provenant de vaches infectes exprimentalement de faon individuelle avec les srovars hardjobovis, copenhageni, grippotyphosa ou canicola ont t prlevs sur une base hebdomadaire pendant plusieurs semaines et ont t assessments avec cette preuve. Au seuil optimal dtermin par la courbe danalyse, la sensibilit et la spcificit relatives de lpreuve taient respectivement de 93,5 % (intervalle de confiance de 95 % = 91,2 % 95,3 %) et 94,7% (intervalle de confiance de 95 % = 93,9 % 95,5 %). Cette preuve permettait de dtecter les anticorps sriques danimaux infects exprimentalement avec le srovar hardjobovis ds la 1resemaine post-inoculation. (Traduit par Docteur Serge Messier) == Introduction == Leptospirosis is usually a zoonotic disease which affects a wide PB-22 range of economically important livestock. The causative organisms belong to the genusLeptospira,which contains over 200 serovars (1). In any given geographic location, only relatively small numbers of serovars are endemic, and these tend to be maintained in specific animal host species (2). In cattle, this disease causes agalactia, abortions, still-births, the birth of weak calves, and possible infertility on a world-wide basis (2). In Canada, theLeptospiraserovars associated with cattle are predominantlyhardjo(now generally recognized ashardjobovis) andpomona,and to lesser extentsgrippotyphosaandicterohaemorrhagiae(310). Currently in Canada, there is a requirement to monitor cattle that are resident in artificial insemination (AI) centres for serovarscanicola, copenhageni(representsicterohaemorrhagiae),grippotyphosa, hardjobovis, pomona,andsejroe.In addition, for the international trade of live cattle, semen, and embryos, many countries request testing for specificLeptospiraserovars, which may include those listed above. The most commonly used and internationally accepted serological test for leptospirosis is the microscopic agglutination test (MAT) (11). However, the MAT has a number of serious problems which indicate the need to develop and implement alternative methods for diagnosing this disease. This study reports the development and evaluation of an indirect enzyme immunoassay capable of detecting bovine antibodies to the 6 serovars of pathogenicLeptospirathat are routinely monitored in Canada. == Materials and methods == == Culture == Leptospira borgpeterseniiserovarshardjobovis(field strain) andsejroe(strain M84);Leptospira interrogansserovarscanicola(strain Hond Utrecht IV),copenhageni(strain M20), andpomona(strain Pomona); andLeptospira kirschneriserovargrippotyphosa(strain Moskva V) were produced at 29C in SPL 5Leptospiramedium (Scientific Protein Laboratories, Waunakee, Wisconsin, USA) that was reconstituted according to the manufacturers directions. Cell counts were determined with a Petroff-Hausser bacteria counter (Canadawide Scientific, Ottawa, Ontario). == The microscopic agglutination test == The MAT was performed in microtitre plates as described (11). PB-22 Live Rabbit polyclonal to JAKMIP1 4-dLeptospiracultures with concentrations adjusted to McFarland Standard #0.5, were used as the antigens. The sera were diluted (serial 2-fold with a final volume of 50 L) in phosphate buffered saline (0.01 M sodium phosphate, 0.145 M sodium chloride, pH 7.2 [PBS]), after which the antigens (50 L) were added. The plates were incubated at room temperature for 1.5 h and then examined by darkfield microscopy. The MAT titre was the reciprocal of the highest dilution of the serum in which 50% of the antigen was agglutinated. == Field sera == Bovine field sera, which were submitted to Canadian Food Inspection Agency (CFIA) regional laboratories (Lethbridge, Alberta; Saskatoon, Saskatchewan; Nepean, Ontario; St. Hyacinthe, Quebec; Sackville, New.