COMPARATIVE INVESTIGATION OF DIFFERENT METHODS FOR THE DETECTION OF INFECTION IN RABBITS CHALLENGED WITH L. interrogans SEROTYPE hardjo

Abstract

Keeping up-to-date with modern diagnostic techniques for leptospirosis as well as continuous improvement of laboratory diagnostic methods resulted in abundant knowledge on the nature and consequences of this infection and its importance in both human and veterinary medicine. In that respect, development and introduction of novel diagnostic tests and procedures have become the paramount issue in the diagnostics of leptospirosis and related infectious diseases. Thus, the goal of this research was to investigate the application of diverse laboratory methods and to evaluate their validity in the diagnostics of leptospirosis. Eleven rabbits were artificially infected with live cultures of L. interrogans serovar hardjo by the method of skin scarification. Blood and blood serum samples of challenged animals were collected every other day throughout the 3-week period (i.e. until day 21), and then once weekly during following five weeks. Blood sera were tested for the presence of L. interrogans serovar hardjo specific antibodies applying the methods of microscopic agglutination (MA) test and ELISA. Blood samples were examined using the method of cultivation in liquid medium by Johnson supplemented with 200μg/1ml 5- fluorouracil (5-FU). Presence/absence of L.interrogans serovar hardjo was confirmed by polymerase chain reaction (PCR) method. In this reaction, a pair of primers separated from the basic structure of the Leptospira interrogans rrs (16S) gene. In MA test, the presence of specific antibodies against L. hardjo in rabbits was confirmed in 67 (36.61%) of 183 investigated sera. Initial positive specific antibody finding was recorded on day 9 post challenge, and it persisted until day 17. In ELISA test, positive and suspect findings were confirmed in 67 and 18 samples, respectively. Initial ELISA-positive finding was observed on day 15, showing increasing tendency throughout the monitoring period and reaching its maximum value on day 42. Method of blood sample cultivation resulted in isolation of L. interrogans serovar hardjo in 33 (18.03%) on day 3 at the earliest, whilst highest isolation rate was observed on day 17 post challenge. Applying polymerase chain reaction (PCR) method, genome or genome sequences of L. interrogans serovar hardjo were detected in 67 (56.30%) out of 119 blood serum samples. PCR method revealed positive finding as early as on day 1 post challenge, whereas the highest rate of positive findings was recorded on day 19. Comparison of the results obtained by methods of cultivation and PCR during the period from experimental day 1 to 21, i.e. period prior to administration of chemotherapeutic agents, demonstrated high level of linear correlation of r = 0.8105 at the 0.01 significance level. After dihydrostreptomycin therapy administered from day 21 post infection, L. interrogans serovar hardjo could not be isolated using the method of blood sample cultivation. Contrary to that, PCR method revealed the presence of L. interrogans serovar hardjo genome in 23 samples.