EQUINE LEPTOSPIROSIS: A STUDY OF LEPTOSPIRA INTERROGANS SEROVAR BRATISLAVA PATHOGENICITY IN HORSES AND DEVELOPMENT OF A DIAGNOSTIC TEST USING REVERSE VACCINOLOGY

Abstract

Leptospirosis is a neglected worldwide zoonotic disease caused by pathogenic gram-negative spirochetes of the genus Leptospira. It can be life threatening for animals and humans. Leptospira can cause livestock losses among pigs, cattle, horses, goats and sheep, thereby inflicting significant losses on the livestock agronomy. This is the first time an analysis using L. interrogans serovar Bratislava, which is believed to be pathogenic for horses, is presented. Six young female foals were challenged with 1 x 109 L. interrogans serovar Bratislava strain PigK151 and observed over a period of 30 days. Temperature, blood and urine, as well as post-mortem samples were evaluated. No pyrexia was noted. PCR was negative from all plasma, urine and tissue samples. No Leptospires were recovered from either plasma or urine culture. All challenged foals developed antibodies against L. interrogans serovar Bratislava as determined by the gold standard microscopic agglutination test (MAT), beginning on day 3 until the last day of the study while all the control animals remained serologically negative. Cross-reactivity on the MAT test results and DNA analysis helped to formulate a reasonable answer for the question about L. interrogans serovar Bratislava pathogenicity in horses. Cross-reactivity among serovars used on the MAT test shows that a better diagnostic test is necessary. Reverse vaccinology has been broadly used to screen for surface-exposed proteins and antigens of important pathogens, like outer membrane proteins and extracellular proteins used as potential vaccine candidates. In order to develop a test that can serve as a diagnostic test and possibly identify the infecting serovar, similar to the gold standard MAT, a reverse vaccinology approach to identify unique proteins among different serovars proved to be a promising strategy. A previous work identified 861 unique proteins among 17 different strains of pathogenic L. interrogans. A subset of these proteins were selected for further study according to their antigenic score, potential B epitopes and size. Eight new leptospiral proteins were cloned, expressed, purified and screened in a serological test along with another four “shared” proteins from L. interrogans. Using these new leptospiral antigens, a new enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for diagnosis of equine leptospirosis. DNA analysis showed that some of these unique proteins (5/8) were only present on reported pathogenic strains and species, suggesting that these proteins might play a role in pathogenicity. Four of the eight new leptospiral antigens presented the very promising results. LA_0711 showed a sensitivity (Se) of 100% and a specificity (Sp) of 69%, LA_1567 results were Se = 98% and Sp = 72%, while LIC11823 values were Se = 98% and Sp = 63% and LEP1GSC077_3193 with Se=97% and Sp = 75%. All antigens but one (WP_061243705), differentiate MAT positive from negative samples (P-value<0.05). Multi-antigen combinations were also tested with the four-most accurate antigens (LA_0711, LA_1567, LIC11823 and LEP1GSC077_3193). ELISA results for these four leptospiral antigens were combined in series and in parallel. In a series approach, a tested sample is positive when it is reactive to all proteins it is tested for. Here, a sensitivity of 95% and specificity of 94% were obtained for series testing for the combination of LA_0711 + LIC11823 + LEP1GSC077_3193. When a parallel method was applied, meaning that a sample considered positive must be reactive to at least one antigen in the combination (combination of two, three or four antigen results), results from LA_1567 and LEP1GSC077_3193 showed the best sensitivity of 99% and specificity of 63%. These single and multi-antigen combinations should be able to supplement if not replace the current MAT for the diagnosis of equine leptospirosis in the near future, after further validation with more equine serum samples that have a more detailed information as clinical phase or identification of infecting serovar/strain done by culture or genetic confirmation.