High virulence in hamsters of four dominant Leptospira serovars isolated from rats in the Philippines

Abstract

Leptospirosis is caused by pathogenic species of Leptospira. The aim of this study was to determine and characterize the pathogenicity of four dominant Leptospira isolates prevailing among rats in the Philippines. The isolates were Leptospira interrogans serovar Manilae strain K64, L. interrogans serovar Losbanos strain K37, L. interrogans serovar Ratnapura strain K5 and Leptospira borgpetersenii serovar Javanica strain K6. Pathogenicities were studied using hamsters, which reproduce severe human leptospirosis. The minimum lethal doses were 10 0 (51) leptospires for K64, K37 and K5, and 10 1 leptospires for K6. Weight loss amongst the Leptospira-infected hamsters was observed from 1 day before death (K64-, K37-and K5-infected hamsters) to as much as 1 week before death for K6-infected hamsters. Similar and varied gross and microscopic lesions were observed amongst infected hamsters, even for strains belonging to the same species (i.e. L. interrogans). The most significant and common histopathological findings were congestion of the glomerulus, disarrangement of hepatic cords and erythrophagocytosis. Other findings were foamy splenic macrophages for K6, severe petechial pulmonary haemorrhage for K64, and hematuria and severe pulmonary congestion for K37. Immunostaining and culture revealed the presence of leptospires in different organs of the infected hamsters. Based on these results, Leptospira isolates from rats in the Philippines were shown to be highly virulent, causing pulmonary haemorrhage, severe hepato-renal damage and death in hamsters even at lower doses. The present findings on experimental leptospirosis support clinical data showing that patients with severe manifestations of leptospirosis, such as pulmonary haemorrhage, are increasing in the Philippines. These findings may serve as a basis to strengthen the early diagnosis and treatment of human leptospirosis.

Characterization of Leptospira infection in suckling and weaning rat pups

“…1B), infected pups were found to have massive hemorrhage or scattered petechiae in their lungs. This was similar to what were also observed in the lungs of Leptospira-susceptible animal models such as hamsters [24] and humans [29].…”

“…Previous reports on experimental leptospirosis mostly focused on acute lethal infection using guinea-pigs [7][8][9], hamsters [2,6,24], and some strains of mice [2,5,[12][13][14]. Rats, which have been well known for its resistance and ability to maintain the leptospires in their kidneys, are considered as resistant animal models of leptospirosis [19].…”

“…These organs were aseptically removed, placed in disposable 2.5 ml syringe, and crushed into 4 ml sterile Korthof's medium with 5-fluorouracil (5-FU; 100 g/ml), and incubated at 30 • C [23,24]. The next day, 500 l of the culture supernatant was sub-cultured in fresh Korthof's medium and further incubated.…”

“…The time immediately after the birth of pups was day 0. 4-, 7-, 14-, and 23-day old pups were then subcutaneously infected with 10 8 , 10 6 , 10 4 and 10 2 organisms of L. interrogans serovar Manilae strain K64 [23,24]. Low passage (<10×) in vitro sub-cultured organisms were suspended in PBS and used for infection [23] (see Section 3.2 for the number of pups per group).…”

“…These organs were fixed in 10% neutral-buffered-formalin (10% NBF), embedded in paraffin, cut in 4 m sections, and stained with haematoxylin-eosin (HE). Immunohistochemistry was performed on paraffin-embedded tissue sections using antiserum against L. interrogans serovar Manilae strain LT 398, which was generated in rabbits [24].…”