山东某规模化鸡场鸡毒支原体的分离鉴定、基因分型、耐药性和致病性分析

Isolation, identification, genotyping, antibiotic resistance, and pathogenicity of Mycoplasma galliscepticum from a chicken farm in Shandong

摘要:

【背景】 鸡毒支原体(Mycoplasma galliscepticum, MG)主要引起家禽慢性呼吸道疾病，不同地区MG菌株的耐药性和致病性差异大，给临床防控增加了难度。【目的】 明确山东省烟台市某规模化鸡场MG分离株的基因分型、耐药情况及致病力，为该地区MG防控提供科学依据。【方法】 从MG阳性鸡群上腭裂拭子分离、纯化获得MG分离株，利用atpG、plsC、mraW、ugpA、DUF3196、lgT和dppC共7个管家基因进行多位点序列分型(multilocus sequence typing, MLST)并构建系统发育树。测定分离株对金霉素、大观霉素、林可霉素、泰乐菌素、泰万菌素、泰妙菌素、沃尼妙林和多西环素的最小抑菌浓度(minimum inhibitory concentration, MIC)，并对相关耐药基因23S rRNA基因V结构域、L4及L22蛋白基因进行测序分析。通过点眼攻毒和气管攻毒两种方式感染28日龄SPF鸡，评价感染10、20和30 d后气囊炎的发病率、气管组织MG载量和病理损伤。【结果】 分离纯化获得9株MG，并首次发现菌株YTX2、YTX5.6、YTX11、YTX12、YTX13、YTX14和YTX15的管家基因plsC和mraW为新的等位基因，ID号分别为33和31，这7株MG分离株属于ST97型，而菌株YTX9和YTX10属于ST16型。所有MG分离株对沃尼妙林和泰妙菌素敏感(MIC≤0.25 μg/mL)，对泰万菌素和多西环素次之(MIC≤4 μg/mL)，对金霉素和林可霉素有一定程度耐药(MIC≥16μg/mL)；属于ST97的7个MG分离株对泰乐菌素存在中等程度耐药(MIC：4-8 μg/mL)；选取菌株YTX5.6、YTX2和YTX10对耐药性相关的23S rRNA基因V结构域、L4和L22蛋白基因测序分析，菌株YTX5.6和YTX2在23S rRNA基因V结构域发生A2069G突变；菌株YTX5.6和YTX2的L4蛋白基因出现G568T突变，在L22蛋白基因上出现T102C、T129C、T252C、C336T和G404A突变；对泰万菌素、泰乐菌素、泰妙菌素、沃尼妙林和多西环素敏感的YTX10株与对照菌株MG R_low株序列一致，未发生突变。在感染后10、20和30 d，菌株YTX5.6气管攻毒组气囊炎发病率分别为80%、60%和80%；菌株YTX10气管攻毒组仅在感染后20 d和30 d出现气囊炎，发病率分别为40%和20%，其余组在整个实验过程中均未发现明显气囊炎。气管攻毒组的鸡在整个感染过程中气管黏膜增厚情况均比点眼攻毒组严重(P<0.05)，菌株YTX5.6气管攻毒组在感染后第20天气管黏膜增厚最严重，平均厚度为300.6 μm。菌株YTX5.6和YTX10气管攻毒方式在感染10、20和30 d后，气管中的MG载量均比菌株YTX5.6和YTX10点眼攻毒方式高。【结论】 本研究从山东省烟台市某规模化鸡群共分离出9株MG菌株，分别属于ST97和ST16基因型，分离株对目前常用抗生素存在不同程度的耐药性，动物感染试验表明不同基因型菌株存在不同致病力。

关键词:鸡毒支原体;耐药性;多位点序列分型;MIC测定;致病性

Abstract:

[Background] Mycoplasma galliscepticum (MG) is the main cause of chronic respiratory diseases in poultry. The different susceptibility to antibiotics and pathogenicity of MG strains from different regions increases the difficulty in the prevention and control of MG infection. [Objective] To clarify the genotypes, antibiotic resistance, resistance gene mutations, and pathogenicity of MG isolates from a chicken farm in Yantai, Shandong, so as to provide a scientific basis for the prevention and control of MG in this area. [Methods] The MG isolates were obtained from the cleft palate swabs of chicken tested positive for MG. Seven housekeeping genes (atpG, plsC, mraW, ugpA, DUF3196, lgT, and dppC) were used for multilocus sequence typing (MLST), and a phylogenetic tree was constructed. The minimum inhibitory concentrations (MICs) of eight antibiotics including chlortetracycline, spectinomycin, lincomycin, tylosin, tylvalosin, tiamulin, valnemulin, and doxycycline against the isolates were determined. The 23S rRNA V domain and L4 and L22 protein genes were sequenced. SPF-grade chickens of 28 days old were infected by eye challenge and trachea challenge, respectively. [Results] Nine MG isolates were obtained in this study, including seven isolates (YTX2, YTX5.6, YTX11, YTX12, YTX13, YTX14, and YTX15) of sequence type 97 (ST97) and two isolates (YTX9 and YTX10) of ST16. New alleles of plsC and mraW were identified in the seven strains of ST97, with the ID of 33 and 31, respectively. All the MG isolates were sensitive to valnemulin and tiamulin (MIC≤0.25 μg/mL), moderately sensitive to tylvalosin and doxycycline (MIC≤4 μg/mL), and resistant to chlortetracycline and lincomycin (MIC≥16 μg/mL). The seven MG isolates of ST97 were moderately resistant to tylosin (MIC: 4-8 μg/mL). The mutation A2069G was identified in the V domain of the 23S rRNA gene in strains YTX5.6 and YTX2. The mutation G568T occurred in the L4 protein gene of strains YTX5.6 and YTX2, and the mutations T102C, T129C, T252C, C336T, and G404A occurred in the L22 protein gene. The strain YTX10 sensitive to tylosin, tylvalosin, tiamulin, valnemulin, and doxycycline showed the same sequence compared with the control strain MG R_low, with no mutation detected. The morbidity of air sacculitis was 80%, 60%, and 80%, respectively, 10, 20, and 30 days after tracheal challenge with the strain YTX5.6. Air sacculitis occurred 20 and 30 days after tracheal challenge with YTX10, with the morbidity of 40% and 20%, respectively. The tracheal mucosal thickening was severer in the tracheal challenge group than in the eye challenge group (P<0.05). Severe tracheal thickening was observed 20 days after tracheal challenge with strain YTX5.6, with an average thickness of 300.6 μm. Tracheal challenge with strain YTX5.6 or YTX10 resulted in significantly higher MG loads than eye challenge with same strain 10, 20, and 30 days post-infection. [Conclusion] In this study, nine MG strains of ST97 and ST16 were isolated from a chicken farm in Yantai, Shandong. The nine isolates demonstrated varied resistance to antibiotics and different pathogenicity to SPF-grade chickens.

Key words:Mycoplasma galliscepticum;antibiotic resistance;multilocus sequence typing;determination of minimum inhibitory concentration;pathogenicity