Pathogenicity of Acinetobacter pittii from beef cattle in mice

doi:10.3969/j.issn.1004-1524.2021.02.06

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (2): 230-238.DOI: 10.3969/j.issn.1004-1524.2021.02.06

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S852.61

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.

Figures/Tables 9

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

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

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.

Fig.2 Clinical symptoms of mice with strain inoculation

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

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.

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×.

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.

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

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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Pathogenicity of Acinetobacter pittii from beef cattle in mice

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