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Detection of Leishmania infantum Infection in Reservoir Dogs Using a Multiepitope Recombinant Protein (PQ10) | ||
| Archives of Razi Institute | ||
| مقاله 4، دوره 75، شماره 3، آذر 2020، صفحه 327-338 اصل مقاله (896.31 K) | ||
| نوع مقاله: Original Articles | ||
| شناسه دیجیتال (DOI): 10.22092/ari.2019.126524.1346 | ||
| نویسندگان | ||
| F. Jameie1؛ A. Dalimi* 1؛ M. Pirestani1؛ M. Mohebali2 | ||
| 1Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran | ||
| 2Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran | ||
| چکیده | ||
| Mediterranean visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum and transmitted by the bites of infected female sand flies. Iran is one of the endemic areas of this disease. Dogs and canines are the major reservoir hosts of Leishmania infantum in the new and old world, including Iran. If visceral leishmaniasis is left untreated, it may result in a 90% mortality rate. The identification and elimination of infected dogs are efficient ways to control this disease. The diagnostic methods used to identify these animals cannot yield 100% detection. Therefore, in the present study, we used a multiepitope recombinant protein (PQ10) to distinguish between symptomatic and asymptomatic infections caused by Leishmania infantum in animal reservoirs (dogs). The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. In the following process, sequencing was performed with plasmid-specific primers and followed by the expression, optimization of expression. The purified recombinant protein was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The efficacy of recombinant PQ10 protein was evaluated by an indirect enzyme-linked immunosorbent assay (ELISA) test using 150 dog sera (25 symptomatic positive sera, 25 asymptomatic positive sera, 80 negative sera, and 20 sera of dogs with other infectious diseases). Direct agglutination test (DAT) as the standard method was used to compare and determine specificity and sensitivity. The results indicated that the sensitivity and specificity of ELISA using infected dog sera were 94% and 86%, respectively. Positive and negative predictive values for dog sera were reported as 87.03% and 93.47%, respectively. This protein was able to identify 92% of asymptomatic dogs with visceral leishmaniasis. The results showed that the recombinant protein PQ10 is able to identify positive cases of canine visceral leishmaniasis, especially asymptomatic cases. | ||
| کلیدواژه‌ها | ||
| Visceral leishmaniasis؛ Leishmania infantum؛ PQ10؛ Recombinant protein؛ Serodiagnosis؛ Dog؛ Iran | ||
| عنوان مقاله [English] | ||
| شناسایی عفونت لیشمانیا اینفانتوم در مخازن سگی با استفاده از یک پروتئین مولتی اپی توپ نوترکیب (PQ10) | ||
| چکیده [English] | ||
| لیشمانیوز احشایی مدیترانهای بیماری زئونوزی است که توسط لیشمانیا اینفانتوم ایجاد و با گزش پشه خاکی ماده آلوده انتقال مییابد. ایران یکی از مناطق اندمیک این بیماری میباشد. سگ و سگ سانان میزبانان مخزن اصلی لیشمانیا اینفانتوم در دنیای جدید و قدیم شامل ایران هستند. در صورتی که بیمار مبتلا به لیشمانیوز احشایی درمان نشود، در 90% موارد منجر به مرگ میشود. از راههای کنترل موثر این بیماری، شناسایی و حذف سگهای آلوده است. روشهای تشخیصی که برای شناسایی این حیوانات استفاده میشوند نمیتوانند 100% آنها را تشخیص دهند. به همین دلیل ما در این تحقیق جهت تشخیص عفونتهای علامتدار و بدون علامت ناشی از لیشمانیا اینفانتوم در مخازن حیوانی (سگ) از پروتئین نوترکیب مولتی اپی توپ PQ10 استفاده نمودیم. توالی کد کننده پروتئین نوترکیب PQ10 در وکتور بیانی pET28 ساب کلون شد و توسط شرکت GENERAY (چین) سنتز گردید. در مرحله بعد، توالی با پرایمرهای اختصاصی پلاسمید تعیین گردید و در نهایت مراحل بیان و بهینه سازی بیان صورت گرفت. پروتئین نوترکیب خالص با استفاده از SDS-PAGE و وسترن بلات مورد تایید قرار گرفت. ارزیابی کارآیی پروتئین نوترکیب PQ10 توسط آزمایش الایزای غیرمستقیم و با به کارگیری سرم 150 سگ (25 سرم مثبت از سگ علامت دار، 25 سرم مثبت از سگ بدون علامت، 80 سرم منفی و 20 سرم سگهای مبتلا به سایر بیماریهای عفونی) انجام شد. جهت مقایسه و تعیین اختصاصیت و حساسیت از آزمایش آگلوتیناسیون مستقیم (DAT) به عنوان روش استاندارد استفاده گردید. نتایج نشان داد که حساسیت و اختصاصیت الایزا با استفاده از سرم سگهای آلوده به ترتیب 94% و 86% است. ارزش اخباری مثبت و منفی در مورد سرم سگی به ترتیب 03/87% و 47/93% محاسبه گردید. این پروتئین قادر به شناسایی 92% از سگهای بدون علامت مبتلا به لیشمانیوز احشایی بود. نتایج نشان میدهد که پروتئین نوترکیب PQ10 قادر به شناسایی موارد مثبت لیشمانیوز احشایی سگی بالاخص موارد بدون علامت میباشد. | ||
| کلیدواژه‌ها [English] | ||
| لیشمانیوز احشایی, لیشمانیا اینفانتوم, PQ10, پروتئین نوترکیب, تشخیص سرولوژیکی, سگ, ایران | ||
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