泰地罗新注射液体外抑菌试验及对感染副猪嗜血杆菌仔猪的保护作用

doi:10.3864/j.issn.0578-1752.2019.16.015

摘要/Abstract

摘要：

【目的】开展了泰地罗新注射液的体外抗菌活性及对仔猪感染副猪嗜血杆菌的保护作用研究。【方法】体外抑菌活性中,采用试管二倍稀释法,以泰拉霉素为对照,测定泰地罗新对副猪嗜血杆菌、胸膜肺炎放线杆菌、多杀性巴氏杆菌、支气管败血性波氏杆菌的最小抑菌浓度（MIC）;在对仔猪感染副猪嗜血杆菌的保护作用中,选择菌量为1×10 ¹⁰ CFU·mL ^-1的副猪嗜血杆菌细菌悬液0.5 mL·kg ^-1bw作为攻毒剂量,进行腹腔注射,复制病理模型。将泰地罗新以2、4、8 mg·kg ^-1bw 3个剂量,泰拉霉2.5 mg·kg ^-1bw单次肌肉注射,治疗人工感染副猪嗜血杆菌的仔猪;在病死猪病料病原菌分离与鉴定中,挑取分离纯化后的副猪嗜血杆菌菌落进行基因组DNA提取,针对副猪嗜血杆菌16sRNA序列设计1对特异性引物,PCR扩增后检验目的DNA片段碱基序列长度。【结果】体外抑菌结果显示,泰地罗新注射液对副猪嗜血杆菌、支气管败血性波氏杆菌、胸膜肺炎放线杆菌及多杀性巴氏杆菌的MIC分别为0.06—8.00、0.06—8.00、0.25—1.00、2.00—32.00 μg·mL ^-1,MIC₅₀分别为0.5、2.0、4.0、0.5 μg·mL ^-1,MIC₉₀为2、8、16、1 μg·mL ^-1,结果表明,泰地罗新注射液对副猪嗜血杆菌、支气管败血性波氏杆菌抑菌效果较强,对胸膜肺炎放线杆菌、多杀性巴氏杆菌抑菌效果较弱;DNA目标片段PCR扩增结果显示,PCR扩增目标片段长度均与文献报道中预测条带一致,特异性片段长度为820bp左右,结果表明,病死猪只均死于副猪嗜血杆菌感染;体内治疗试验结果显示,泰地罗新注射液低、中、高剂量组增重分别为（2.5±0.2）、（2.9±0.2）、（2.9±0.3）kg,死亡率分别为40%、0、0,有效率分别为40%、100%、100%,治愈率分别为40%、100%、100%;泰拉霉素注射液组增重为（3.0±0.2）kg,死亡率为10%,有效率为90%,治愈率为80%;感染不给药组增重为（2.1±0.1）kg,死亡率为70%。结果表明,泰地罗新注射液高、中剂量组与泰拉霉素对照药物组无显著性差异（P>0.05）,均能迅速的减轻临床症状,具有显著的治疗效果。泰地罗新低剂量组治疗效果与感染不给药组相当,无显著性差异（P>0.05）。【结论】泰地罗新注射液按4 mg·kg ^-1bw临床推荐给药剂量,可有效治疗由副猪嗜血杆菌感染引起的猪呼吸道疾病。

关键词: 泰地罗新注射液, MIC, 副猪嗜血杆菌, 仔猪

Abstract:

【Objective】The study was carried out to investigate the antibacterial activity in vitro and protection of tildipirosin injection against artificially infected Haemophilus Paracoides in piglets. 【Method】Minimal inhibitory concentration (MIC) of Tildipirosin injection against Haemophilus paracoides, Actinobacillus pleuropneumonia, Pasteurella multiflora, and Bronchial septicemic wave bacillus were determined by two-fold dilution method in vitro. In the protective effect of tildipirosin injection against Haemophilus paracoides in piglets, 0.5 mL·kg ^-1bw suspension of Haemophilus paracoides with a bacterial volume of 1 10 ¹⁰ CFU·mL ^-1 was selected as the challenge dose for intraperitoneal injection to replicate the pathological model. All the therapeutic groups of pigs were medicated via intramuscular injection in single dose with tildipirosin injection (2, 4, 8 mg·kg ^-1bw), tulathromycin (2.5 mg·kg ^-1bw). In the process of isolating and identifying dead pig pathogens, isolated and purified Haemophilus paracoides colonies were selected for genomic DNA extraction. A pair of specific primers were designed for the 16s RNA sequence of Haemophilus paracoides, and the base sequence length of the target DNA fragment was tested after PCR amplification.【Result】The results of the antibacterial activity in vitro showed that MIC of Haemophilus paracoides, Septicopharynx, Actinobacillus pleuropneumoniae and Pasteurella multocida for tildipirosin injection was 0.06-8, 0.06-8, 0.25-1, and 2-32 μg·mL ^-1, respectively, which indicated the effect of Tildipirosin injection against Haemophilus paracoides and Septicopharynx was stronger than Actinobacillus pleuropneumoniae and Pasteurella multocida. PCR amplification results of the DNA target fragment showed that the length of the PCR amplification target fragment was consistent with the predicted band, and the specific fragment length was about 820bp, which indicated that all the dead pigs died of Haemophilus paracoides infection. The result of protective effect indicated that, in tildipirosin injection groups (2, 4, 8 mg·kg ^-1bw), the average gain of weight was (2.5±0.2), (2.9±0.2), (2.9±0.3) kg, respectively; the mortality rate were 40%, 0 and 0, respectively; the effective rate was 40%, 100% and 100%, respectively; the cure rate was 40%, 100% and 100%, respectively. In tulathromycin injection groups (2.5 mg·kg ^-1bw), the average gain of weight was (3.0±0.2) kg; the mortality rate were 10%; the effective rate was 90%; the cure rate was 80%. In infective control group, the average gain of weight was (2.1±0.1) kg; the mortality rate were 70%. The results of in vitro treatment showed that there was no significant difference between the high- and medium-dose group of tildipirosin and the tulathromycin group (P>0.05), which could alleviate the clinical symptoms rapidly and had significant therapeutic effects. The therapeutic effect of tildipirosin low-dose group was comparable to the non-administered group, and there was no significant difference (P>0.05). 【Conclusion】Through the above results, the product was recommended to be administered at a dose of 4 mg·kg ^-1bw, which could effectively treat respiratory diseases in pigs caused by Haemophilus Parasuis infection.

Key words: tildipirosin injection, MIC, Haemophilus Paracoides, piglet

李国基, 闫超群, 麻雨桥, 谢顺, 顾欣, 曹莹, 黄士新, 黄显会. 泰地罗新注射液体外抑菌试验及对感染副猪嗜血杆菌仔猪的保护作用[J]. 中国农业科学, 2019, 52(16): 2899-2911.

LI GuoJi, YAN ChaoQun, MA YuQiao, XIE Shun, GU Xin, CAO Ying, HUANG ShiXin, HUANG XianHui. Antibacterial Activity in Vitro and Protection of Tildipirosin Injection Against Artificially Infected Haemophilus Paracoides in Piglets[J]. Scientia Agricultura Sinica, 2019, 52(16): 2899-2911.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.16.015

https://www.chinaagrisci.com/CN/Y2019/V52/I16/2899

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组别 Groups	数量（头） Numbers (Head)	体重 Weights (kg)	剂量 Dosage (mg·kg^-1bw)	给药方案 Drug regimen
健康对照组 Healthy control group	10	12.5±0.6	—	不感染,不给药 No infection, No administration
泰地罗新注射液高剂量组 High dose group of Tildipirosin injection	10	12.9±0.9	8.0	感染,肌注注射 Infection,im
泰地罗新注射液中剂量组 Middle dose group of Tildipirosin injection	10	12.9±1.1	4.0	感染,肌注注射 Infection,im
泰地罗新注射液低剂量组 Low dose group of Tildipirosin injection	10	12.7±0.9	2.0	感染,肌注注射 Infection,im
泰拉霉素注射液 Tulathromycin injection	10	13.1±0.5	2.5	感染,肌注注射 Infection,im
感染对照组 Infective control group	10	12.5±0.6	—	感染,不给药 Infection, No administration

体系组分 System components	用量 The dosage (μL)
灭菌超纯水 Sterilized ultra-pure water	12.6
10×PCR buffer	2
dNTP（2.5 mmol·L^-1）	1.6
上游引物 Forward primer	0.8
下游引物 Reverse primer 模板DNA（Template DNA）	0.8 2
rTag DNA酶 rTag DNA enzyme, 5 U·μL^-1	0.2

菌株 Bacterial strain	日龄接种 Inoculate days	活菌量 Living bacterium quantity (CFU/mL)	接种剂量 Inoculation dosage (mL·kg^-1）	数量（头） Number (Head)	攻毒结果 Poison attack results
菌株 Bacterial strain	日龄接种 Inoculate days	活菌量 Living bacterium quantity (CFU/mL)	接种剂量 Inoculation dosage (mL·kg^-1）	数量（头） Number (Head)	发病数量（头） Morbidity（Head）	死亡数量（头） Mortality（Head）
13R	7-8周龄 7-8 weeks	1×10¹⁰	0.3	10	5	1
		1×10¹⁰	0.5	10	10	7
		1×10¹⁰	0.8	10	10	10
		1×10¹⁰	1	10	10	10
TSB培养基 TSB medium	7-8周龄 7-8 weeks	1×10¹⁰	1mL	10	0	0

时间 Time (h)	健康对照组 Healthy control group	泰地罗新注射液高剂量组 High dose group of Tildipirosin injection	泰地罗新注射液中剂量组 Middle dose group of Tildipirosin injection	泰地罗新注射液低剂量组 Low dose group of Tildipirosin injection	泰拉霉素注射液 Tulathromycin injection	感染对照组 Infective control group
0	39.2±0.18	39.4±0.19	39.1±0.22	39.2±0.20	38.9±0.22	39.3±0.31
1	39.1±0.19	40.2±0.18	40.6±0.23	40.4±0.22	40.2±0.18	40.5±0.29
3	39.2±0.22	41.2±0.21	41±0.19	41.1±0.21	41.2±0.21	41±0.22
4	38.6±0.21	40.9±0.20	40.8±0.17	41.3±0.22	41.1±0.19	41.8±0.28
6	39.3±0.20	40.4±0.19	40.4±0.16	40.7±0.24	40.7±0.23	41.2±0.17
8	39.1±0.17	40.1±0.23	40±0.21	40.5±0.18	40.3±0.21	40.8±0.19
12	38.9±0.20	39.5±0.18	39.5±0.22	40.2±0.19	39.6±0.18	40.9±0.24
24	39±0.19	39.3±0.23	39.5±0.18	40.2±0.22	39.2±0.19	40.6±0.22
36	39.2±0.23	39.4±0.25	39.1±0.19	39.5±0.22	39.4±0.27	40.5±0.28
48	38.8±0.16	39.2±0.21	39.3±0.20	39.5±0.24	39.1±0.26	40.7±0.26
60	39.1±0.21	39.2±0.20	39±0.20	39.4±0.22	38.9±0.29	40.8±0.21
72	38.9±0.22	38.9±0.17	39.4±0.22	39.3±0.22	39.2±0.19	40.6±0.27
84	39.2±0.18	39.3±0.22	39.4±0.25	38.9±0.21	39±0.18	40.5±0.30
96	39.1±0.19	38.8±0.20	38.9±0.21	39.2±0.22	39.3±0.21	40.4±0.22
108	39.3±0.20	39.1±0.24	39.2±0.19	39.1±0.19	38.9±0.21	40.5±0.22
120	38.7±0.22	39.1±0.22	39±0.17	39.4±0.22	39.2±0.22	40.4±0.24
132	39.2±0.18	39.2±0.19	39.2±0.20	38.8±0.24	39.4±0.19	40.3±0.25
144	39.1±0.21	39.2±0.21	39.3±0.20	39.2±0.21	39.1±0.21	40.4±0.21
156	39.4±0.21	39.4±0.25	39.2±0.18	39±0.22	39.2±0.18	40.3±0.24

组别 Groups	试验前体重 Pretrial weight (kg)	试验后体重 Posttest weigh (kg)	增重 Weight increment (kg)
健康对照组 Healthy control group	12.5±0.6	15.5±0.7	3.0±0.2a
泰地罗新高剂量组 High dose group of Tildipirosin injection	12.9±0.9	15.8±1.2	2.9±0.3a
泰地罗新中剂量组 Middle dose group of Tildipirosin injection	12.9±1.1	15.7±1.2	2.9±0.2a
泰地罗新低剂量组 Low dose group of Tildipirosin injection	12.7±0.9	15.2±0.9	2.5±0.2b
泰拉霉素注射液 Tulathromycin injection	13.1±0.5	16.1±0.6	3.0±0.2a
感染对照组 Infective control group	12.5±0.6	14.6±0.7	2.1±0.1c