Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine

Abstract

Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 10⁴ Leptospira/mL, and urine with pH ≤ 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 °C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.

Keywords: EMJH-STAFF; Icterohaemorrhagiae; Leptospira; PBS; isolation; pH; swine urine.

Figure 1

Experiment 1: Process of culture preparation (a) in EMJH medium, (b) in EMJH-STAFF medium and (c) with filtration of each urine sample.

Figure 2

Experiment 2: Steps of preparation of seven dilutions of pure culture (10⁹ Leptospira/mL) in each of ten selected urine samples before transfer into culture medium (EMJH-STAFF).

Figure 3

Number of (a) EMJH and (b) EMJH-STAFF cultures from spiked swine urine samples with the microscopic (dark field 200×) evaluation results at week one, two, three and four post inoculation differentiated into the following categories: □ no Leptospira/no contamination, ▤ contamination, ▨ Leptospira plus contamination and ■ pure Leptospira. ★ significant relationship between the result category at the corresponding week of evaluation and culture (EMJH and EMJH-STAFF), (p < 0.01).

Figure 4

Percentage of □ EMJH and ■ EMJH-STAFF cultures from spiked swine urine samples with pH ≤ 7 and > 7 and microscopically (dark field, 200×) detectable Leptospira with and without contamination at (a) week one, (b) week two, (c) week three and (d) week four post inoculation. ★ significant relationship between urine sample pH and detectability of Leptospira in cultures at the corresponding week of evaluation (p < 0.01).

Figure 5

Mean absolute Leptospira copy numbers per mL (log₁₀) as estimated by qPCR in EMJH and EMJH-STAFF cultures with Leptospira copy number increase and decrease ■ 2 days (n = 30 EMJH cultures; n = 30 EMJH-STAFF cultures) compared to □ 28 days (n = 14 EMJH cultures; n = 30 EMJH-STAFF cultures) post inoculation.

Figure 6

Number of microscopic (dark field, 200×) evaluations including week one, two, three and four post inoculation of (a) ten EMJH and (b) ten EMJH-STAFF cultures from spiked swine urine samples stored for 24 h at 4 °C □ without and ■ with added buffer (PBS) and differentiated into the following categories: no Leptospira/no contamination, contamination, Leptospira plus contamination and pure Leptospira.