Research on the Virulence Identification and Preservation Methods of Desert-type Leishmania donovani Strains

doi:10.12300/j.issn.1674-5817.2023.034

Abstract

Abstract:

Objective To determine the virulence of desert-type Leishmania donovani strains through animal infection experiments and to explore preservation methods for maintaining their pathogenicity. Methods The isolated strain was cultured in vitro for 7, 30, 36, 44, 60, 90, and 150 days, respectively， and inoculated into Lagurus lagurus (L.lagurus) with the dose of 2.6×10⁵ per animal by intraperitoneal injection. The spleen coefficient, infection rate, and antibody positive rate of the inoculated animals were detected at day 60 after infection. The desert-type Leishmania donovani strain was further inoculated with Cricetulus migratorius (C.migratorius) and L. lagurus, respectively, for passaging and preservation. The survival time of two kinds of animals and pathogenicity change of the stain in their bodies were compared. Results After inoculation of desert-type Leishmania donovani strains cultured in vitro for 7-150 days, the spleen coefficient of inoculated L.lagurus gradually increased from 1% on day 7 to 2.2% on day 30, which was more than 10 times of the normal spleen coefficient. Additionally, on day 60, the spleen coefficient remained 3 times higher than the normal value. The infection rate and antibody positive rate decreased from 80% on day 7 to 0% on day 60. At 90 days, there were no significant differences between the infected groups and the control group, and all the observed indexes were within the normal range. The survival time of L.lagurus infected with the in vivo passage strain ranged from 1 to 13 months, and half of the infected individuals died within 4 months. In contrast, C.migratorius had a survival time ranging from 5 to 31 months, and half of the infected individuals died within an average of 13.7 months. There was a significant difference in the average time of death between the two groups (t=0.000 1, P<0.001), but no significant difference in spleen coefficient (t=0.990, P>0.05). This strain exhibited equal virulence in both animals and remained virulent for up to 4 years after continuous passage. Conclusion With the prolonged culture time, the virulence of the strain decreases gradually. At 90 d, it has no pathogenicity to L. lagurus. Long-term in vitro culture fails to preserve it's pathogenicity to L.lagurus. Only in vivo inoculation can maintain the virulence of this strain.

Key words: Kala-azar, Desert-type Leishmania donovani, Virulence, Maintenance of virulence, Lagurus lagurus, Cricetulus migratorius

CLC Number:

R382

Lifu LIAO, Yun LUO, Shen SHI, Yimei XU. Research on the Virulence Identification and Preservation Methods of Desert-type Leishmania donovani Strains[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 619-625.

Figures/Tables 4

Figure 1 The effect on infection rate and antibody positive rate of desert-type L.donovani on different culture time in L.lagurus

Figure 2 Effect of desert-type L.donovani strain incubation time in vitro on spleen coefficient of L.lagurus

Table 1 Infection results of L.lagurus and C.migratorius that were inoculated with desert-type Kala-azar strains through

样本 Sample	存活/月 Survival/month	死亡数量/只 Number of deaths n	濒死时间/月 Death time/month	脾脏系数/% Spleen coefficient/%
草原兔尾鼠 L.lagurus	1~6	21	4.19±1.47	1.17±0.92
	7~9	13	7.92±0.86	2.60±2.52
	10~13	7	11.43±0.98	2.46±2.18
灰仓鼠 C.migratorius	5~9	7	6.71±1.60	1.18±1.40
	10~18	15	13.67±2.74	1.86±1.29
	19~31	21	22.95±2.91	1.92±1.71
草原兔尾鼠合计 Total of L.lagurus	1~13	41	6.61±3.02	1.84±1.88
灰仓鼠合计 Total of C.migratorius	5~31	43	17.07±6.80^a	1.78±1.52^b

Figure 3 Pathology results of spleen on L.lagurus （A） and C.migratorius （B） by desert-type L.donovani strain （Giemsa stain）Note：The blank arrows show amastigote （Leishman-donovan body） of desert-type L.donovani strain.

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