Study animal
In a dairy herd in southern Chile, eleven out of 130 calves died after presenting clinical signs such as depression and haematuria. One of these calves, a female of eight months, was submitted to the Department of Animal Pathology at the Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile for necropsy.
Pathological findings
Necropsy showed a marked yellowing pigmentation in all mucosal body openings and in the subcutaneous tissue, fat and muscles. There were also isolated petechia in the kidneys and the bladder contained approximately two liters of red-tinged urine (Figure 1).
A urine sample was collected by puncturing the bladder and sent to the Leptospirosis and Paratuberculosis Laboratory, Department of Biochemistry and Microbiology, at the Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile. All the procedures were in strict accordance with the recommendations in the Guide of Use of Animals for Research of Universidad Austral de Chile, approved by the Committee on the Ethics of Animals for Research (www.uach.cl/direccion/ investigacion/uso_animales.htm).
Bacteriological analysis
The urine sample was investigated using dark field microscopy and bacterial structures consistent with Leptospira were found. Thereafter, 200 μl of urine sample with four replicates was cultured in EMJH medium at 29°C [8]. After a month of incubation, a positive result was reported with a typical Dinger ring growth.
Leptospira DNA was extracted from positive cultures and in order to identify Leptospira species, primers covering the most common pathogenic Leptospira species were used (664–665 L. kirschneri fla gene; 1280–1281 L. interrogans IS1500; 1805–1809 L. borgpetersenii IS1533) [9]. The total PCR reaction was 50 μl, of which 5 μl was 10× Taq polymerase buffer (Promega, Madison, WI), 2 μl dNTPs (2.5 mM stock containing all four dNTPs) (Promega, Madison, WI), 0.5 U Taq Polymerase (Promega, Madison, WI), 1 μl (each) primer (stock concentration = 100 pmol/μl; final concentration 2 pmol/μl), 35.5 μl dH2O and 5 μl template. The PCR reactions considered 40 cycles of 94°C for 15 sec; 60°C for 30 sec and 68°C for 2 min. then 10°C hold. Negative and positive PCR controls were included as well as DNA extraction negative and positive controls.
To refine our understanding of the Leptospira specie and serovar associated with this clinical case, a Variable Number Tandem Repeat (VNTR) analysis was done. The VNTR primers were designed exclusively for use with L. interrogans [10-12]. PCR products for VNTR loci 4, 7, 10, 23, 27, 29, 30, 31, and 36 were assessed using the same PCR reaction as described above and the primers used were as previously reported [12]. PCR products were separated by agarose gel electrophoresis and visualized, and their sizes were calculated by comparing with reference standards (100-bp ladder; Invitrogen, Carlsbad, CA) and with the literature [10,11]. As a complement, we also amplified the gene secY, which is a house keeping gene that consists of alternating conserved and variable regions, making it suitable to deduce primers that generate amplicons with sufficient sequence heterogeneity to enable phylogenetic interpretation for Leptospira [13]. A 202 bp product was amplified by conventional PCR in 25 μl mixture containing 5 μl diluted template (1:100), 0.2 μM each primers SecYIVF (5′-GCGATTCAGTTTAATCCTGC-3′) and SecYIV (5′-GAGTTAGAGCTCAAATCTA-AG-3′), 0.625 U GoTaq Flexi DNA Polymerase in 1X Green Buffer GoTaq (Promega, Madison, WI), 3.0 mM MgCl2, 0.3 mM dNTPs (Promega, Madison, WI), and 400 ng mL-1 bovine serum albumin (BSA; BioLabs, Ipswich, England). Cycle conditions included an initial denaturation step at 95°C for 5 min followed by 40 cycles at 94°C for 1 min, 57°C for 1 min and 72°C for 1 minute and a final elongation step at 72°C for 10 minutes. The PCR products obtained were separated on 1.5% agarose gel, stained with Gel Red (GelRed, Biotium Inc, Hayward, U.S), excised and purified using a commercial kit (E.Z.N.A® Gel Extraction Kit, Omega Bio-Tek, Norcross, U.S). Amplicons were sequenced by Macrogen Inc (Seoul, Korea). The consensus nucleotide sequence obtained in this study was compared with secY gene of Leptospira interrogans serovar Hardjo prajitno (GenBank accession number EU357983.1). DNA alignments were done using clustalW tools (http://www.ebi.ac.uk/Tools/msa/clustalw2).
