牛源皮特不动杆菌对小鼠的致病性分析

doi:10.3969/j.issn.1004-1524.2021.02.06

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (2): 230-238.DOI: 10.3969/j.issn.1004-1524.2021.02.06

牛源皮特不动杆菌对小鼠的致病性分析

樊利虹¹(), 郭红瑞¹, 吴江², 易军³, 马晓平¹, 苟丽萍¹, 谢跃¹, 叶刚¹, 左之才¹^,^*()

1.四川农业大学动物医学院,四川温江 611130
2.深圳海关动植物检验检疫技术中心,广东深圳 518045
3.四川省畜牧科学研究院,四川成都 610066

收稿日期:2020-08-03 出版日期:2021-02-25 发布日期:2021-02-25
通讯作者: 左之才
作者简介:*左之才,E-mail: zzcjl@126.com
樊利虹(1996—),女,四川成都人,硕士研究生,研究方向为中西兽医与临床。E-mail: 2323702149@qq.com
基金资助:
四川省科技计划(2018NZ0002);四川省科技计划(2019YFQ0012);国家重点研发计划(2018YFD0501800);国家现代农业产业技术体系四川肉牛创新团队项目(SCCXTD-2020-13)

Pathogenicity of Acinetobacter pittii from beef cattle in mice

FAN Lihong¹(), GUO Hongrui¹, WU Jiang², YI Jun³, MA Xiaoping¹, GOU Liping¹, XIE Yue¹, YE Gang¹, ZUO Zhicai¹^,^*()

1. College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China
2. Inspection and Quarantine Center for Animals and Plants, Shenzhen Customs, Shenzhen 518045, China
3. Sichuan Academy of Animal Husbandry, Chengdu 610066, China

Received:2020-08-03 Online:2021-02-25 Published:2021-02-25
Contact: ZUO Zhicai

摘要/Abstract

摘要：

为探究来源于牛呼吸道疾病综合征发病牛的皮特不动杆菌对小鼠的致病性,对其进行相关毒力基因的检测和小鼠致病性试验,试验内容主要包括临床症状观察、病理剖检、组织病理学观察,以及半数致死量(LD₅₀)和各器官载菌量的测定。结果显示,牛源皮特不动杆菌ZZCNC1807-6和ZZCSF1807-9菌株对小鼠均具有较强的致死性,对小鼠的LD₅₀分别为4.487×10⁷ CFU·mL^-1和3.177×10⁸ CFU·mL^-1。剖检死亡小鼠可见,牛源皮特不动杆菌主要造成小鼠皮下充血、肺淤血肿胀、肠道积气积液等症状,可引起心、肝、脾、肺、肾、脑等多种器官感染。组织病理学观察显示,小鼠各器官病变均较轻微,主要表现在肝、肺中有少量炎性细胞浸润,脾中白髓细胞坏死和纤维增生,其中ZZCNC1807-6攻菌组的病变较ZZCSF1807-9攻菌组相对严重。载菌量分析结果显示,2株菌对小鼠肝、心、脾、肺和肾的侵袭力差异不大,但ZZCNC1807-6对脑的侵袭力强于ZZCSF1807-9。综上,ZZCNC1807-6菌株的毒力强于ZZCSF1807-9。

关键词: 皮特不动杆菌, 毒力基因, 半数致死量, 组织病理学

Abstract:

In order to explore the pathogenicity of two strains of Acinetobacter pittii from bovine respiratory disease complex (BRDC) infected beef cattle to mice, the related virulence genes were detected and the pathogenicity test of mice was carried out, which mainly included clinical symptom observation, pathological examination, histopathological observation, and determination of median lethal dosage (LD₅₀) and bacterial load in various organs. The results showed that ZZCNC1807-6 and ZZCSF1807-9 stains were highly lethal to mice, and their LD₅₀ reached 4.487×10⁷ CFU·mL^-1 and 3.177×10⁸ CFU·mL^-1, respectively. After pathology autopsy, it was seen that Acinetobacter pittii mainly caused subcutaneously hyperemia, lung congestion and swelling, intestinal pneumatosis and effusion and other symptoms in mice. And it could cause infection of many organs, such as heart, liver, spleen, lung, kidney and brain. Histopathological observation showed that the pathological changes of various organs in mice were mild, mainly manifested in a small amount of inflammatory cells infiltration in liver and lung, and necrosis and fibrous hyperplasia of white pulp cells in spleen. Among them, the pathological changes of mice in ZZCNC1807-6 group were more serious than those in ZZCSF1807-9 group. The analysis of bacterial load showed that the invasiveness of the two strains to the liver, heart, spleen, lung and kidney of mice had little difference, but ZZCNC1807-6 strain had stronger invasiveness to the brain than ZZCSF1807-9. To sum up, the virulence of strain ZZCNC1807-6 was stronger than that of ZZCSF1807-9.

Key words: Acinetobacter pittii, virulence genes, median lethal dose, histopathology

中图分类号:

S852.61

樊利虹, 郭红瑞, 吴江, 易军, 马晓平, 苟丽萍, 谢跃, 叶刚, 左之才. 牛源皮特不动杆菌对小鼠的致病性分析[J]. 浙江农业学报, 2021, 33(2): 230-238.

FAN Lihong, GUO Hongrui, WU Jiang, YI Jun, MA Xiaoping, GOU Liping, XIE Yue, YE Gang, ZUO Zhicai. Pathogenicity of Acinetobacter pittii from beef cattle in mice[J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 230-238.

图/表 9

表1 皮特不动杆菌毒力基因检测的PCR引物序列

Table 1 Primers sequence of virulence gene of Acinetobacter pittii

目的基因 Target gene	引物序列(5'→3') Primer sequence (5''→3')	产物长度 Product length/bp	退火温度 Annealing temperature/℃
ompA	F:CGCTTCTGCTGGTGCTGAAT R:CGTGCAGTAGCGTTAGGGTA	531	50
bfmS	F:TTGCTCGAACTTCCAATTTATTATAC R:TTATGCAGGTGCTTTTTTATTGGTC	1 428	53
kpsMT Ⅱ	F:GCGCATTTGCTGATACTGTTG R:TCCAGACGATAAGCATGAGCA	272	63
cnf1	F:AAGATGGAGTTTCCTATGCAGGAG R:TTCAGAGTCCTGCCCTCATTATT	498	63
cnf2	F:AATCTAATTAAAGAGAAC R:CATGCTTTGTATATCTA	543	63
bap	F:TACTTCCAATCCAATGCTAGGGAGGGTACCAATGCAG R:TTATCCACTTCCAATGATCAGCAACCAAACCGCTAC	1 562	65
epsA	F:AGCAAGTGGTTATCCAATCG R:ACCAGACTCACCCATTACAT	451	50
plcD	F:GCGCTTATTGGTGGGCGCAAT R:CTGAACGGTGGCTTGTTGATAATG	246	55
lpxM	F:CAACCCATTCAATTTGGGCG R:GGTCATTGATGTGAATTGAGC	363	55
feoA	F:GTTAGGGTTTCCCACAAGGGC R:AGGATCGGATGCGGTGCGTT	315	55
bl ${a_{PER -}}_{1}$	F:ATGAATGTCATTATAAAAGC R:AATTTGGGCTTAGGGCAAGAAA	910	50

表1 皮特不动杆菌毒力基因检测的PCR引物序列

Table 1 Primers sequence of virulence gene of Acinetobacter pittii

目的基因 Target gene	引物序列(5'→3') Primer sequence (5''→3')	产物长度 Product length/bp	退火温度 Annealing temperature/℃
ompA	F:CGCTTCTGCTGGTGCTGAAT R:CGTGCAGTAGCGTTAGGGTA	531	50
bfmS	F:TTGCTCGAACTTCCAATTTATTATAC R:TTATGCAGGTGCTTTTTTATTGGTC	1 428	53
kpsMT Ⅱ	F:GCGCATTTGCTGATACTGTTG R:TCCAGACGATAAGCATGAGCA	272	63
cnf1	F:AAGATGGAGTTTCCTATGCAGGAG R:TTCAGAGTCCTGCCCTCATTATT	498	63
cnf2	F:AATCTAATTAAAGAGAAC R:CATGCTTTGTATATCTA	543	63
bap	F:TACTTCCAATCCAATGCTAGGGAGGGTACCAATGCAG R:TTATCCACTTCCAATGATCAGCAACCAAACCGCTAC	1 562	65
epsA	F:AGCAAGTGGTTATCCAATCG R:ACCAGACTCACCCATTACAT	451	50
plcD	F:GCGCTTATTGGTGGGCGCAAT R:CTGAACGGTGGCTTGTTGATAATG	246	55
lpxM	F:CAACCCATTCAATTTGGGCG R:GGTCATTGATGTGAATTGAGC	363	55
feoA	F:GTTAGGGTTTCCCACAAGGGC R:AGGATCGGATGCGGTGCGTT	315	55
bl ${a_{PER -}}_{1}$	F:ATGAATGTCATTATAAAAGC R:AATTTGGGCTTAGGGCAAGAAA	910	50

图1 毒性基因检测结果 A,bap基因;B,plcD基因;C,lpxM基因;D,feoA基因;E,ompA基因;F,epsA基因。M,DL2000 DNA分子质量标准;1,阴性对照;2~6分别为菌株ZZCNC1807-4、ZZCNC1807-6、ZZCNC1807-7、ZZCJL1807-8、ZZCSF1807-9。

Fig.1 Test result of virulence genes A, bap gene; B, plcD gene; C, lpxM gene; D, feoA gene; E, ompA gene; F, epsA gene. M, DL2000 DNA marker; 1, Negative control; 2-6 represented strain ZZCNC1807-4, ZZCNC1807-6, ZZCNC1807-7, ZZCJL1807-8, ZZCSF1807-9, respectively.

图2 攻菌组小鼠的临床症状

Fig.2 Clinical symptoms of mice with strain inoculation

表2 供试菌株对SPF级昆明小鼠的感染试验结果

Table 2 Test result of strains to SPF Kunming mice

组别 Group	ZZCNC1807-6			ZZCSF1807-9
组别 Group	感染活菌浓度 Concentration of infected bacteria/(CFU·mL^-1)	注射剂量 Infection dose/mL	死亡率 Mortality rate/%	感染活菌浓度 Concentration of infected bacteria/(CFU·mL^-1)	注射剂量 Infection dose/mL	死亡率 Mortality rate/%
1	3.2×10⁸	0.2	100	1.6×10⁹	0.2	100
2	1.6×10⁸	0.2	83.4	8×10⁸	0.2	83.3
3	8×10⁷	0.2	66.7	4×10⁸	0.2	66.7
4	4×10⁷	0.2	50.0	2×10⁸	0.2	33.3
5	2×10⁷	0.2	33.3	1×10⁸	0.2	0
6	1×10⁷	0.2	0	0.5×10⁸	0.2	0
对照Control	0	0.2	0	0	0.2	0

图3 病理剖检结果 A1,对照组小鼠各器官无明显病变;B1,ZZCNC1807-6攻菌组小鼠皮下充血,肠道积气积液;C1,ZZCSF1807-9组小鼠皮下充血,肠道积气积液;A2,对照组小鼠肺无明显异常现象;B2,ZZCNC1807-6攻菌组小鼠肺严重淤血;C2,ZZCSF1807-9攻菌组小鼠肺表现为局部淤血、肿胀。

Fig.3 Results of pathological examination A1, No obvious lesion in each organ of the control group mice; B1, The mice in ZZCNC1807-6 group had subcutaneous congestion and intestinal pneumatosis and effusion; C1, The mice in ZZCSF1807-9 group were subcutaneously congested, as intestinal wall became thinner, and intestinal gas accumulated and fluid accumulated; A2, No obvious lesion in lungs of the control group mice; B2, The lungs of mice in ZZCNC1807-6 group showed severe pulmonary congestion; C2, The lungs of mice in ZZCSF1807-9 group showed local congestion and swelling.

图4 小鼠的组织病理学观察 1,肺;2,肝;3,脾;4,心;5,肾;A,对照组;B,ZZCNC1807-6攻菌组;C,ZZCSF1807-9攻菌组;a,中性粒细胞;b,淋巴细胞;c,血管白细胞边缘化;d,肝细胞空泡变性;e,坏死细胞;f,纤维增生。放大倍数400倍。

Fig.4 Histopathological observation of mice 1, Lung; 2, Liver; 3, Spleen; 4, Heart; 5, Kidney; A, Control group; B, ZZCNC1807-6 group; C, ZZCSF1807-9 group; a, Neutrophils; b, Lymphocytes; c, Marginalization of vascular leukocytes; d, Hepatocyte vacuolation; e, Necrotizing cells; f, Fibrous hyperplasia. Magnification power was 400×.

图5 小鼠器官的分离菌培养结果 A1~A2,对照组;B1~B2,ZZCNC1807-6攻菌组;C1~C2,ZZCSF1807-9攻菌组。a,肝;b,脾;c,肾;d,脑;e,心;f,肺。

Fig.5 Results of bacteria isolation from mice organs A1 and A2, Control group; B1 and B2, ZZCNC1807-6 group; C1 and C2, ZZCSF1807-9 group。a, Liver; b, Spleen; c, Kidney; d, Brain; e, Heart; F, Lung.

图6 分离菌株的革兰氏染色结果(1 000×) A,ZZCNC1807-6攻菌组;B,ZZCSF1807-9攻菌组。

Fig.6 Gram staining results of isolated strains (1 000×) A, ZZCNC1807-6 group; B, ZZCSF1807-9 group.

表3 ZZCNC1807-6和ZZCSF1807-9菌株接种小鼠各脏器的载菌量

Table 3 Bacterial load in various organs of mice inoculated with ZZCNC1807-6 and ZZCSF1807-9 CFU·g-1

脏器Organ	ZZCNC1807-6	ZZCSF1807-9
肝Liver	5.46×10⁷	1.61×10⁸
心Heart	2.90×10⁷	1.52×10⁷
脾Spleen	5.18×10⁸	3.26×10⁸
肺Lung	1.02×10⁸	2.02×10⁸
肾Kidney	1.38×10⁸	9.55×10⁷
脑Brain	1.53×10⁸	2.70×10⁵

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牛源皮特不动杆菌对小鼠的致病性分析

Pathogenicity of Acinetobacter pittii from beef cattle in mice

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