Akibekov Orken S, Gajimuradova Aissarat M, Syzdykova Alfiya S, Zhumalin Aibek Kh, Zhagipar Fariza S, Tosini Fabio, Akanova Zhannara Zh, Gubaidullin Nurtai N, Askarova Nasipkhan A
Department of Microbiology and Biotechnology, Faculty of Veterinary and Livestock Technology, Saken Seifullin Kazakh Agrotechnical Research University, 62 Zhenis Avenue, Astana 010000, Kazakhstan.
European Union Reference Laboratory for Parasites, lstituto Superiore di Sanità, 299, Viale Regina Elena, Roma 00161, Italy.
Vet World. 2024 Sep;17(9):2124-2135. doi: 10.14202/vetworld.2024.2124-2135. Epub 2024 Sep 20.
Diagnosis of trichinellosis at the intestinal stage during larval development is the primary challenge in the early detection and treatment of trichinellosis. The use of serine protease as a diagnostic marker for serological tests has been the subject of various studies, but data on serine protease in the intestinal phase are still insufficient for a proper diagnosis. This study aimed to establish the duration of the intestinal phase for early diagnosis and to determine the level of expression of the serine protease gene in and larvae.
We used European isolates from pigs and larvae isolated from spontaneously infected wild carnivorous animals (wolf, Karaganda region) in Central Kazakhstan. Isolation of larvae from the meat of infected animals was carried out using the compressor method. For two species of , 36 mice (in each group 18 mice) were infected with 250 larvae and euthanized by intramuscular injection of xylazine followed by an intravenous overdose of anestofol at 3, 5, 7, 14, 21, and 30 dpi (each day 3 infected mice) and one control group (3 mice). Sequencing and bioinformatics methods were used to determine the DNA and cDNA of the serine protease gene, and molecular methods (DNA extraction, reverse transcription polymerase chain reaction, and sequence) were used to measure the accumulation of serine protease transcripts in isolated larvae.
The results showed differences in the duration of intestinal phase between and . The intestinal larvae of were observed from 7 to 30 dpi, and the intensity of invasion increased up to 30 dpi (p < 0.001), while in the case of , the increase in larval growth in the intestinal phase decreased to 21 dpi, and only an increase of 1.6 ± 0.88 (p < 0.01) was detected at 30 dpi. muscle larvae were detected at 21 dpi, compared with at 14 dpi. This characteristic was also reflected in the levels of serine protease transcripts in the samples. Accumulation was observed in both cases higher in the muscular stage of development, whereas the duration of the intestinal stage of made it possible to detect serine protease at 30 dpi.
The intestinal stage of lasts for 30 days, indicating that the use of serine protease is useful for the identification of intestinal infection. Furthermore, this protein can be used to identify and in laboratory samples. Serine protease can be used as a marker for serological diagnosis. Within the framework of the research topic, it is important to conduct further studies on the species specificity of the obtained recombinant protein. It is necessary to focus on identifying highly specific proteins for early disease detection.
旋毛虫病在幼虫发育的肠道阶段的诊断是旋毛虫病早期检测和治疗的主要挑战。使用丝氨酸蛋白酶作为血清学检测的诊断标志物一直是各种研究的主题，但关于肠道阶段丝氨酸蛋白酶的数据仍不足以进行准确诊断。本研究旨在确定肠道阶段的持续时间以进行早期诊断，并确定丝氨酸蛋白酶基因在和幼虫中的表达水平。
我们使用了来自猪的欧洲分离株以及从哈萨克斯坦中部卡拉干达地区自然感染的野生食肉动物（狼）体内分离出的幼虫。采用压缩法从感染动物的肉中分离幼虫。对于两种旋毛虫，将36只小鼠（每组18只小鼠）感染250条幼虫，并在感染后第3、5、7、14、21和30天（每天3只感染小鼠）通过肌肉注射甲苯噻嗪，随后静脉注射过量的安乐福进行安乐死，并设置一个对照组（3只小鼠）。使用测序和生物信息学方法确定丝氨酸蛋白酶基因的DNA和cDNA，并使用分子方法（DNA提取、逆转录聚合酶链反应和测序）测量分离幼虫中丝氨酸蛋白酶转录本的积累。
结果显示和的肠道阶段持续时间存在差异。在感染后第7至30天观察到的肠道幼虫，侵袭强度在第30天之前增加（p < 0.001），而对于，肠道阶段幼虫生长的增加在第21天下降，在第30天仅检测到1.6±0.88的增加（p < 0.01）。在感染后第21天检测到肌肉幼虫，而在第14天检测到。这一特征也反映在样本中丝氨酸蛋白酶转录本的水平上。在发育的肌肉阶段，两种情况下均观察到积累增加，而的肠道阶段持续时间使得在第30天能够检测到丝氨酸蛋白酶。
的肠道阶段持续30天，表明使用丝氨酸蛋白酶有助于识别肠道感染。此外，这种蛋白质可用于在实验室样本中鉴定和。丝氨酸蛋白酶可作为血清学诊断的标志物。在该研究主题的框架内，有必要对获得的重组蛋白的物种特异性进行进一步研究。有必要专注于鉴定用于早期疾病检测的高度特异性蛋白质。
