Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus

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Abstract

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil.

Introduction

Leptospirosis is a zoonotic infection of global importance caused by pathogenic spirochetes belonging to the genus Leptospira. Synanthropic rodents are the most important maintenance hosts, but several other animal species (e.g., wild, domestic, and farm animals) can act as significant reservoirs of leptospires. The transmission of bacteria to new hosts usually occurs through indirect contact of the skin (particularly skin abrasions) or mucous membranes with contaminated water and soil or through direct contact with urine or tissues of infected animals [1], [2], [3], [4].

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine for some time after infection and can transmit the pathogen from animal to animal or animal to human [1]. Therefore, livestock farming plays an important role as a major occupational risk factor for human leptospirosis. In addition, bovine leptospirosis constitutes a serious economic problem for the livestock industry, causing abortions, reduced milk yield, mortality in calves, and decreased daily weight gain [5].

Leptospirosis in cattle is most frequently the result of infection with serovar Hardjo (L. borgpetersenii subtype Hardjobovis or Leptospira interrogans subtype Hardjo-prajitno) [6], [7]. However, other serovars may be associated with leptospiral infection, including Icterohaemorrhagiae, Bratislava, Pomona, Canicola, and Grippotyphosa [1], [7]. Consequently, the commercial vaccines that have been produced against bovine leptospirosis are cellular suspensions containing one or more of these serovars [7], [8]. Similarly to vaccine formulations, the reference method for serological diagnosis of leptospirosis (microscopic agglutination test, MAT) is based on a collection of representative strains of Leptospira, in which serum samples are reacted with live antigen suspensions to verify serovar-specific antibody levels [9]. In this context, surveillance and monitoring systems for detection of new Leptospira strains are important approaches for the control of leptospirosis. Here, we report the isolation of an L. interrogans serogroup Australis serovar Muenchen from a bovine foetus and bacterial characterization by immunological and molecular techniques.

Section snippets

Isolation and leptospiral culture

The renal tissue from a stillborn bovine foetus (R2) was removed and macerated under sterile conditions in liquid Ellinghausen–McCullough–Johnson–Harris (EMJH) medium without antibiotics, supplemented with Leptospira Enrichment EMJH (Difco, BD Diagnostics, Sparks, MD, USA). The samples were incubated at 30 °C and examined weekly (during 90 days) by dark-field microscopy. The foetal tissues were collected at the slaughterhouse during a routine cattle slaughter in Aceguá City (31°45″, 54°3″W), Rio

Results

Molecular characterization by VNTR typing with seven discriminatory primers revealed the presence of tandem repeats. According to the dendrogram proposed by Majed et al. [10], the isolate belongs to species L. interrogans serogroup Australis, serovar Muenchen (Fig. 1). After isolation and VNTR typing, a phenotypic analysis using mAbs against leptospiral membrane-associated proteins was performed. Viable intact leptospires were attached to slide chambers coated with poly-l-lysine, and mAbs bound

Discussion

VNTR typing has proved to be a highly powerful and discriminatory method for investigating bacterial genetic diversity in general, and for differentiating serogroups and serovars from Leptospira. However, VNTR is not able to discriminate between L. interrogans serovars Icterohaemorrhagiae and Copenhageni. In contrast, the use of only one VNTR locus, VNTR19 or VNTR23, is sufficient for discriminating serovars Muenchen, Jalna, and Bratislava [10]. Thus, the primer set used was sufficient for

Conclusions

The VNTR identified a L. interrogans serovar Muenchen isolated from a bovine foetus. The Leptospira isolated expressed important virulence factors (LipL32 and Ligs) and caused clinical signs compatible with the leptospirosis in hamster model. Reference sera presented co-agglutinating antibodies against the Aceguá isolate, suggesting the presence of new strain in the geographic area among bovines. The use of Aceguá isolate as antigen on MAT battery can contribute to the epidemiological

Acknowledgments

This study was supported by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ).

References (16)

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These authors contributed equally to the experimental work.

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