Abstract: Objective　To investigate the changes in the prevalence of Babesia microti infections, spleen morphology and proportions of splenic immune cells in BALB/c mice following intravenous and intraperitoneal injections, so as to provide insights into unraveling the immune regulatory mechanisms of Babesia infections.  Methods　Laboratory⁃maintained B. microti strains were prepared into whole blood samples with 10% prevalence of B. microti infection. A total of 75 BALB/c mice were randomly divided into three groups, including the normal control group, intravenous injection group, and intraperitoneal injection group, of 25 mice in each group. Mice in the intravenous and intraperitoneal injection groups were administered 100 μL of whole blood samples with 10% prevalence of B. microti infection, with the day of injection recorded as d0, and animals in the normal control group were given no treatments. Blood was sampled from mice in each group via the tail tip on d7, d14, d21, d28 and d35, and prepared into thin⁃film blood smears, and B. microti infection was observed in red blood cells. Five mice were randomly sampled from each group and sacrificed on d7, d14, d21, d28 and d35, and spleen was collected for measurement of spleen size and weight. In addition, splenic cells were isolated, and the proportions of CD3e+ T cells, CD45R+ B cells, CD49b+ nature killer (NK) cells, and F4/80+ macrophages were detected in CD45+ lymphocytes using flow cytometry.  Results　 The prevalence of B. microti infection in the intravenous (22.80%) and intraperitoneal injection groups (44.82%) peaked on d7 ([χ2] = 8.141, P < 0.01) and then rapidly decreased, and no parasites were observed on d35. The longest mouse spleen length [(32.91 ± 2.20) mm] and width [(9.82 ± 0.43) mm], and the greatest weight [(0.78 ± 0.10) g] were found on d14 in the intravenous injection group, and the longest spleen length [(32.42 ± 3.21) mm] and width [(10.25 ± 0.73) mm], and the greatest weight [(0.73 ± 0.09) g] were seen in the intraperitoneal injection group on d21, d7 and d14, respectively. There were significant differences among the intravenous injection group, intraperitoneal injection group and the normal control group in terms of spleen length (F = 10.310, P < 0.05), width (F = 9.824, P < 0.05), and weight (F = 10.672, P < 0.05) on d21, and the mouse spleen length, width and weight were all significantly greater in the intraperitoneal injection group than in the intravenous injection group (all P values < 0.05). The proportions of splenic CD3e+ T cells [(60.60 ± 6.20)% and (39.68 ± 7.62)%], CD45R+ B cells [(43.32 ± 2.08)% and (49.53 ± 4.90)%], CD49b+ NK cells [(6.88 ± 1.34)% and (7.71 ± 1.59)%], and F4/80+ macrophages [(2.21 ± 0.29)% and (3.80 ± 0.35)%] peaked on d14, d21, d21 and d14 in the intravenous and intraperitoneal injection groups, respectively. There were significant differences in the proportions of CD3e+ T cells (F = 16.730, P  < 0.05) and F4/80+ macrophages (F = 15.941, P  < 0.05) among the intravenous injection group, intraperitoneal injection group and normal control group on d14, and a higher proportion of CD3e+ T cells and a lower proportion of F4/80+ macrophages were detected in the intravenous injection group than in the intraperitoneal injection group (both P values < 0.01). There were significant differences among the intravenous injection group, intraperitoneal injection group and normal control group on d21 in terms of proportions of splenic CD3e+ T cells (F = 9.252, P < 0.05), CD45R+ B cells (F = 14.349, P  < 0.05), CD49b+ NK cells (F = 13.436, P  < 0.05), and F4/80+ macrophages (F = 8.180, P  < 0.05), and a higher proportion of CD3e+ T cells and lower proportions of CD45R+ B cells and F4/80+ macrophages were detected in the intravenous injection group than in the intraperitoneal injection group (all P values < 0.01). In addition, there was a significant difference in the proportion of CD3e+ T cells among the intravenous injection group, intraperitoneal injection group and normal control group on d28 (F = 9.772, P < 0.05), and a lower proportion of CD3e+ T cells was found in the intravenous injection group than in the intraperitoneal injection group (P < 0.01). Conclusions　Both intraperitoneal and intravenous routes are effective to induce B. microti infections in BALB/c mice, and the prevalence of B. microti infections is higher in BALB/c mice through the intraperitoneal route than through the intravenous route. Intraperitoneal and intravenous injections with B. microti cause diverse spleen morphologies and proportions of splenic immune cells in mice, indicating routes of B. microti infections cause different impacts on immune response mechanisms in mice. 

Key words: Babesia microti, Intraperitoneal injection, Intravenous injection, Spleen, Immune cell；Mice 

摘要： 目的　观察经静脉和腹腔接种田鼠巴贝虫后，BALB/c小鼠田鼠巴贝虫染虫率及脾形态和脾免疫细胞占比变化，为巴贝虫感染免疫调控机制相关研究提供参考。方法　利用实验室保种田鼠巴贝虫虫株制备染虫率为10%的全血血样，将75只BALB/c小鼠随机分为正常对照组、静脉接种组和腹腔接种组，每组25只。腹腔接种组和静脉接种组小鼠分别经腹腔和静脉接种染虫率为10%的全血100 μL，接种当天记为d0；正常对照组为健康小鼠，不作任何处理。分别于d7、d14、d21、d28、d35采集各组小鼠尾尖血样制作薄血膜血片，观察并比较红细胞染虫情况。此外，分别于d7、d14、d21、d28、d35从各组随机选择5只小鼠，剖杀后取脾脏，测量脾脏大小和质量；分离脾细胞，使用流式细胞仪检测脾脏CD3e+ T细胞、CD45R+B细胞、CD49b+自然杀伤细胞（natural killer cell，NK细胞）和巨噬细胞（Mø细胞）（F4/80）等免疫细胞占CD45+ 淋巴细胞的比例。结果　静脉接种组和腹腔接种组小鼠染虫率均于d7达到高峰，染虫率分别为22.80%、44.82%，差异有统计学意义（[χ2]  = 8.141，P < 0.01）；随后两组染虫率均迅速下降，至d35时血涂片中已无法观察到虫体。静脉接种组小鼠脾脏长度［（32.91 ± 2.20） mm］、宽度［（9.82 ± 0.43） mm］及质量［（0.78 ± 0.10） g］均于d14达最大值，腹腔接种组小鼠脾脏长度［（32.42 ± 3.21） mm］、宽度［（10.25 ± 0.73） mm］及质量［（0.73 ± 0.09） g］分别在d21、d7、d14达最大值。d21时，静脉接种组、腹腔接种组、正常对照组小鼠脾脏长度、宽度和质量差异均有统计学意义（F = 10.310、9.824、10.672，P均< 0.05）；腹腔接种组小鼠脾脏长度、宽度和质量均大于静脉接种组（P 均< 0.05）。静脉接种组和腹腔接种组小鼠脾脏CD3e+ T细胞 ［（60.60 ± 6.20）% 和（39.68 ± 7.62）%］、CD45R+B细胞 ［（43.32 ± 2.08）%）和（49.53 ± 4.90）%］、CD49b+NK细胞 ［（6.88±1.34）% 和（7.71±1.59）%］、Mø细胞（F4/80）占比［（2.21 ± 0.29）% 和（3.80 ± 0.35）%］分别于d14、d21、d21、d14达峰值。d14时，静脉接种组、腹腔接种组、正常对照组小鼠脾脏CD3e+ T细胞和Mø细胞（F4/80）占比差异均有统计学意义（F =16.730、15.941，P均< 0.05）；静脉接种组小鼠脾脏CD3e+ T细胞占比高于腹腔接种组，Mø细胞（F4/80）占比低于腹腔接种组（P 均< 0.01）。d21时，3组小鼠脾脏CD3e+ T细胞、CD45R+ B细胞、CD49b+ NK细胞和Mø细胞（F4/80）占比差异均有统计学意义（F = 9.252、14.349、13.436、8.180，P均< 0.05）；静脉接种组小鼠脾脏CD3e+ T细胞占比高于腹腔接种组，CD45R+ B细胞和Mø细胞（F4/80）占比均低于腹腔接种组（P 均< 0.01）。d28时，3组小鼠脾脏CD3e+ T细胞占比差异有统计学意义（F = 9.772，P < 0.05）；静脉接种组CD3e+ T细胞占比低于腹腔接种组（P  < 0.01）。结论　腹腔接种和静脉接种均可致BALB/c小鼠感染田鼠巴贝虫，且腹腔接种后小鼠染虫率高于静脉接种。腹腔接种和静脉接种田鼠巴贝虫对小鼠脾脏形态和免疫细胞占比造成的影响存在差异，提示不同接种方式对小鼠免疫应答机制具有不同影响。

关键词: 田鼠巴贝虫, 腹腔接种, 静脉接种, 脾脏, 免疫细胞, 小鼠