荒漠型杜氏利什曼原虫虫株在体内外的致病力及保存方法研究

doi:10.12300/j.issn.1674-5817.2023.034

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (6): 619-625.DOI: 10.12300/j.issn.1674-5817.2023.034

荒漠型杜氏利什曼原虫虫株在体内外的致病力及保存方法研究

廖力夫¹()(), 罗芸², 史深¹, 徐艺玫¹()()

^1.新疆维吾尔自治区疾病预防控制中心, 乌鲁木齐 830002
^2.乌鲁木齐地窝堡机场海关, 乌鲁木齐 830013

收稿日期:2023-03-15 修回日期:2023-07-08 出版日期:2023-12-25 发布日期:2024-01-06
通讯作者: 徐艺玫（1973—），女，硕士，研究员，研究方向：实验动物管理。E-mail： 545763836@qq.com。ORCID： 0000-0003-0013-4044
作者简介:廖力夫（1957—），男，学士，研究员，研究方向：野生动物驯化，黑热病疫源地及控制研究。E-mail： liaolif@sina.com。ORCID： 0009-0005-0402-1524
基金资助:
国家自然科学基金项目“新疆塔里木盆地荒漠型黑热病保虫宿主调查研究”(30460120);“喀什绿洲黑热病多传染源多媒介多病原调查”(30760217)

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

Lifu LIAO¹()(), Yun LUO², Shen SHI¹, Yimei XU¹()()

^1.Xinjiang Center for Disease Control and Prevention, Urumqi 830002, China
^2.Urumqi Diwobao Airport Customs of the People's Republic of China, Urumqi 830013, China

Received:2023-03-15 Revised:2023-07-08 Published:2023-12-25 Online:2024-01-06
Contact: XU Yimei (ORCID: 0000-0003-0013-4044), E-mail: 545763836@qq.com

摘要/Abstract

摘要：

目的通过动物攻毒实验观察荒漠型杜氏利什曼原虫虫株在动物体内外的致病力，探索保持该虫株致病力的保存方法。方法将从rK39抗体阳性灰仓鼠脾脏中分离获得的荒漠型杜氏利什曼原虫虫株分别在体外培养基中传代培养至7 d、30 d、36 d、44 d、60 d、90 d和150 d后，按2.6×10⁵条/只剂量腹腔接种至草原兔尾鼠，接种后60 d计算动物的脾脏系数、虫株感染率和抗体阳性率。进一步将荒漠型杜氏利什曼原虫虫株分别接种灰仓鼠和草原兔尾鼠进行传代保种，比较两种动物感染该虫株后的存活时间和致病力变化。结果体外培养7～150 d的荒漠型杜氏利什曼原虫虫株接种后，草原兔尾鼠的脾脏系数由7 d的1.0%上升至30 d的2.2%，达到正常脾脏系数（0.15%）的10倍以上，而60 d的脾脏系数虽有下降，但仍然达正常值的3倍；虫株感染率和抗体阳性率由7 d的80%逐步下降至60 d的0%；90 d时的各项观测指标与对照组相比无明显差异，均在正常值范围内。传代感染虫株后，草原兔尾鼠的存活时间为1～13个月，感染的半数个体于接种后4个月内死亡；而灰仓鼠的存活时间为5～31个月，感染的半数个体于接种后13.7个月内死亡；两种动物的平均死亡时间差异显著（t=0.000 1，P<0.001），脾脏系数差异无统计学意义（t=0.990，P>0.05）。该虫株在两种动物体内的致病力一致，且在动物体内连续传代4年仍然具有致病力。结论荒漠型杜氏利什曼原虫虫株随体外培养时间延长，其致病力逐步降低，90 d时对草原兔尾鼠已无致病力，说明培养基传代培养方法不能保持该虫株对动物的致病力。动物体内传代接种才可以保持该虫株对动物的致病力。

关键词: 黑热病, 荒漠型杜氏利什曼原虫, 致病力, 毒力维持, 草原兔尾鼠, 灰仓鼠

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

中图分类号:

R382

廖力夫, 罗芸, 史深, 徐艺玫. 荒漠型杜氏利什曼原虫虫株在体内外的致病力及保存方法研究[J]. 实验动物与比较医学, 2023, 43(6): 619-625.

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.

图/表 4

图1 荒漠型杜氏利什曼原虫虫株体外培养时间对草原兔尾鼠感染率和抗体阳性率的影响

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

图2 荒漠型杜氏利什曼原虫虫株体外培养时间对草原兔尾鼠脾脏系数的影响

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

表1 荒漠型黑热病虫株体内传代接种草原兔尾鼠和灰仓鼠的感染结果 (passagein vivo)

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

图3 草原兔尾鼠（A）和灰仓鼠（B）脾脏感染利什曼原虫的印片染色结果比较（Giemsa染色）注：图中黑色箭头指示利什曼原虫无鞭毛体。

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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荒漠型杜氏利什曼原虫虫株在体内外的致病力及保存方法研究

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

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