Mukhtar Masham, Ghafoor Aamir, McClelland Michael, Akhtar Fareeha, Rasheed Muhammad Adil
University Diagnostic Laboratory, Institute of Microbiology, University of Veterinary and Animal Science, Lahore, Pakistan.
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, United States.
Front Microbiol. 2024 Nov 13;15:1467230. doi: 10.3389/fmicb.2024.1467230. eCollection 2024.
This study involves the development and molecular characterization of the isogenic markerless knockout mutant SG Δ, a genetically engineered live attenuated strain aimed at controlling Gallinarum (SG) infection in poultry. The mutant was generated by deleting the gene using -Red recombination technology, impairing adenylosuccinate lyase, necessary for purine biosynthesis. An 1,180 bp deletion was engineered within the gene, leaving a residual 298 bp genomic scar resulting in a purine auxotrophic mutant. Phenotypically, SG Δ showed a 66.5% reduction in growth in LB broth compared to the wild-type strain and failed to grow in minimal media without adenosine. Growth was restored to near wild-type levels with 0.3 mM adenosine supplementation, demonstrating the strain's conditional attenuation. pathogenicity assessments revealed that oral inoculation of SG Δ into 3-day-old chickens at a dose of 2 × 10 CFU resulted in zero mortality, compared to an 80% mortality rate in chickens challenged with the wild-type strain. The SG Δ strain exhibited significantly reduced clinical signs and lesion scores, with clinical sign scores dropping from 2.5/3 with the wild-type to 0.4/3 with the Δ mutant, and lesion scores decreasing from 2.9/3 to 0.3/3. Additionally, the mutant was efficiently cleared from liver and spleen tissues by 14 days post-inoculation, unlike the wild-type strain, which persisted until the experiment's end on day 21. The SG Δ mutant shows potential as a safe alternative for preventing fowl typhoid, highlighting the promise of targeted genetic attenuation in developing effective vaccines for poultry diseases.
本研究涉及同基因无标记敲除突变体SG Δ的构建及其分子特征分析，该突变体是一种基因工程减毒活菌株，旨在控制家禽感染鸡伤寒沙门氏菌（SG）。该突变体是通过使用λ - Red重组技术缺失基因而产生的，该基因缺失会损害嘌呤生物合成所必需的腺苷酸琥珀酸裂解酶。在该基因内设计了一个1180 bp的缺失，留下298 bp的基因组残余片段，从而产生嘌呤营养缺陷型突变体。从表型上看，与野生型菌株相比，SG Δ在LB肉汤中的生长减少了66.5%，并且在没有腺苷的基本培养基中无法生长。添加0.3 mM腺苷后，生长恢复到接近野生型水平，证明了该菌株的条件性减毒。致病性评估显示，以2×10⁷ CFU的剂量给3日龄鸡口服接种SG Δ，死亡率为零，而用野生型菌株攻击的鸡死亡率为80%。SG Δ菌株的临床症状和病变评分显著降低，临床症状评分从野生型的2.5/3降至Δ突变体的0.4/3，病变评分从2.9/3降至0.3/3。此外，与野生型菌株不同，野生型菌株在实验第21天结束时仍持续存在，而该突变体在接种后14天就从肝脏和脾脏组织中被有效清除。SG Δ突变体显示出作为预防禽伤寒安全替代物的潜力，突出了靶向基因减毒在开发家禽疾病有效疫苗方面的前景。
