Farklı Hayvan Türlerinde Atığa Sebep Olan Leptospirozisin Moleküler Epidemiyolojisi
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Dosyalar
Tarih
2023
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Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Leptospirozis, hastalığın etkeni olan Leptospira spp.'nin patojenitesine ve konakçıya özel faktörlere göre insanlarda ve hayvanlarda abort, nefritis, hepatitis, üveitis, plasentitise sebep olabilen zoonoz bir hastalıktır. Etken; insanlar ve evcil hayvanların birbirleriyle doğrudan veya enfekte idrarla teması sonucu bulaşabilmektedir. Mikroskobik aglütinasyon testi (MAT), enfeksiyonun teşhisinde yaygın kullanılmakla beraber testin önemli bazı dezavantajları bulunmaktadır. Bu sebeple, enfeksiyonun teşhisine yönelik hızlı, kolay, ekonomik, güvenilir ilave metotlara ihtiyaç vardır. Bu çalışmada; atık yapan hayvanların numunelerinde patojen ve saprofitik Leptospira spp. varlığının araştırılması, etkenin izole edilmesi, rekombinant LipL32 (rLipL32) ve rLigA proteinlerinin güncel, hızlı bir yöntem olan In Vitro Assembly (IVA) klonlama metodu ile elde edilmesi, idrar numunelerinde etken spesifik IgG, IgA, IgM varlığının rLipL32 ve rLigA temelli indirekt ELISA ile taranması, pre-adsorpsiyon ile testin güvenilirliğinin araştırılması amaçlandı. Bu çalışma iki adımlı olarak gerçekleştirildi. İlk adımda; ülkemizin 7 farklı coğrafi bölgesindeki çiftliklerden 2019-2021 yılları arasında teşhis amacıyla laboratuvarımıza gelen atık yapan sığır, koyun atlara ait toplamda 287 adet numune kullanıldı. DNA izolasyonu yapılan numuneler PZR ile 16srRNA, LipL32, cimA genleri yönünden değerlendirildi. 287 numuneden etkenin izolasyonu için EMJH besiyeri kullanıldı. İkinci adımda; Almanya Leipzig Üniversitesi Veteriner Fakültesi Laboratuvarına 2022-2023 yılları arasında gelen sığır ve atlara ait 175 adet numune kullanıldı. Leptospira interrogans serovar Hardjo'nun LigA ve LipL32 genleri pQE30 ekspresyon vektörüne aktarıldı. E. coli M15'e transforme edildikten sonra klonlama işlemi PZR ve sekans analizi ile doğrulandı. Rekombinant proteinler SDS-PAGE ve Western Blot yöntemleri değerlendirildi. İdrar ve serum numunelerindeki anti-Leptospira IgG'lerin Western Blot ile gösterilmesinde ve anti-Leptospira IgG, IgM, IgA'lerin ELISA yöntemi ile değerlendirilmesinde rLipL32 ve rLigA proteinleri kullandı. Cut-off değerinin belirlenmesinde total protein miktarı düşük sığır idrar numuneleri kullanıldı. Geliştirilen ELISA yönteminin güvenilirliğinin arttırılması amacıyla idrar numunelerindeki olası E.coli'ye özgü antikorları ortadan kaldırmak için numuneler önceden adsorbe edildi. Pre-adsorpsiyon öncesi ve sonrası ELISA sonuçları karşılaştırıldı.PZR sonuçları değerlendirildiğinde; toplam 143 (% 49,83) numunenin 16srRNA yönünden pozitif olduğu belirlendi. Bunlar içerisinde sığırların %44,64'ü 16srRNA, %26,79'u LipL32, %4,02 cimA geni; koyunların %48,39 16SrRNA, %25,81'i LipL32, %16,13 cimA geni, atların %87,5'i 16SrRNA geni yönünden pozitif diğer iki gen yönünden negatif bulundu. PZR pozitif numunelerden sadece 3 adet taze idrardan izolasyon gerçekleştirilmekle beraber bunlardan 2 tanesi patojen Leptospira spp. idi. Anti-Leptospira immunglobulinleri Western Blot ve ELISA yöntemleri ile sığır ve atların idrar, serum numunelerinde tespit edildi. Sığır idrar ELISA sonuçları; rLipL32 ELISA; %36,71 IgG, %38,28 IgM, %29,68 IgA yönünden pozitif bulunurken rLigA ELISA; %46,88 IgG, %25,78 IgM ve %28,90 IgA yönünden pozitif bulundu. İdrar örneklerinin ön adsorpsiyonu ELISA'nın özgüllüğünü arttırmasına rağmen sonuçları önemli ölçüde değiştirmedi. Pre-adsorpsiyon öncesi rLipL32 ile %44,53 rLigA ile %46,09 iken pre-adsorpsiyon sonrası rLipL32 ile %42,97 rLigA ile %45,31 oranında yüksek özgüllük ile pozitiflik belirlenmesine rağmen aynı sığırlara ait kan serumlarından MAT negatif sonuçlar alındı. Sonuç olarak; ülkemizde leptospirozisin yaygınlığının arttığı, göz ardı edilmemesi gerektiği, rLipL32 ve rLigA temelli geliştirilen indirekt ELISA yönteminin idrar numunelerinde spesifik IgG, IgA, IgM analizinde kullanılabileceği, etkenin izolasyonunda ise en uygun numunenin taze idrar olabileceği kanaatine varıldı.
Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp. It can cause abortion, nephritis, hepatitis, uveitis and placentitis in humans and animals. The agent can be transmitted by humans and pets through direct contact or infected urine. Although microscopic agglutination test (MAT) is widely used in the diagnosis of infection, the test has some disadvantages. Therefore, additional fast, easy, economical and reliable methods for diagnosis are needed. This study aimed to investigate the presence of pathogenic and saprophytic Leptospira spp. in samples of aborted animals by PCR, isolating the agent, obtaining recombinant leptospiral proteins by the In Vitro Assembly (IVA) cloning method for the screening of agent-specific IgG, IgA, IgM in urine samples with indirect ELISA. In the first step of the study a total of 287 samples of cattle, sheep and horses from farms in seven different geographical regions of our country, collected between 2019 and 2021, were investigated. DNA was isolated from the samples and evaluated by PCR for 16srRNA, lipL32 and cimA gene of Leptospira spp.. EMJH medium was used for cultivation and enrichment of the agent from the 287 samples. In the second step 175 urine and serum samples of cattle and horses, collected between 2022 and 2023, were screened for Leptospira-specific immunoglobulins in the Laboratory of the Faculty of Veterinary Medicine of the University of Leipzig, Germany. In detail, ligA and lipL32 genes of Leptospira interrogans serovar Hardjo were cloned into the pQE30 expression vector. Recombinant proteins were evaluated by SDS-PAGE and Western Blot. rLipL32 and rLigA proteins were used to detect anti-Leptospira IgGs in urine and serum samples by Western Blot and to evaluate anti-Leptospira IgG, IgM, IgA by ELISA. Bovine urine samples with low total protein amount were used for cut-off value calculation. In order to increase the reliability of the developed ELISA method, urine samples were pre-adsorbed to eliminate possible E. coli-specific antibodies. ELISA results before and after pre-adsorption were compared. A total of 143 (49.83%) samples were determined positive by 16srRNA PCR. In detail, 44.64%, 48.39% and 87.5% of the cattle, sheep and horse samples were positive for 16srRNA gene, respectively. Furthermore, 26.79% and 25.81% of the cattle and sheep samples were positive for lipL32, respectively. cimA gene was detected in 4.02% of cattle and 16.13% of the sheep samples. lipL32 and cimA could not be detected in horse samples. Only 3 out of 287 cultural analyzed samples were positive for Leptospira shown by 16srRNA PCR. These samples were fresh urine samples. Two of them were PCR positive for the pathogenic Leptospira gene lipL32. Anti-Leptospira immunoglobulins were detected in urine and serum samples of cattle and horses by Western Blot and ELISA. In the bovine urine ELISA with rLipL32 36.71%, 38.28% and 29.68% of the samples were determined positive for IgG, IgM and IgA, respectively. In the ELISA with rLigA, 46.88%, 25.78% and 28.90% were positive for IgG, IgM and IgA, respectively. Pre-adsorption of the urine samples increased the specificity of the ELISA, but did not change significantly the results. Before pre-absorption, 44.53% and 46.09% of the samples were positive in the rLipL32 and rLigA ELISA, respectively. After pre-absorption, 42.97% and 45.31% of the samples were positive in the rLipL32 and rLigA ELISA, respectively. ELISA was positive although there were MAT negative results from blood sera from the same cattle. It can be concluded: I) that the prevalence of leptospirosis has increased in our country and should not be ignored, II) that the developed indirect ELISA method, based on rLipL32 and rLigA, can be used in the analysis of specific IgG, IgA, IgM in urine samples and III) that the most suitable sample for the isolation of Leptospira is fresh urine.
Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp. It can cause abortion, nephritis, hepatitis, uveitis and placentitis in humans and animals. The agent can be transmitted by humans and pets through direct contact or infected urine. Although microscopic agglutination test (MAT) is widely used in the diagnosis of infection, the test has some disadvantages. Therefore, additional fast, easy, economical and reliable methods for diagnosis are needed. This study aimed to investigate the presence of pathogenic and saprophytic Leptospira spp. in samples of aborted animals by PCR, isolating the agent, obtaining recombinant leptospiral proteins by the In Vitro Assembly (IVA) cloning method for the screening of agent-specific IgG, IgA, IgM in urine samples with indirect ELISA. In the first step of the study a total of 287 samples of cattle, sheep and horses from farms in seven different geographical regions of our country, collected between 2019 and 2021, were investigated. DNA was isolated from the samples and evaluated by PCR for 16srRNA, lipL32 and cimA gene of Leptospira spp.. EMJH medium was used for cultivation and enrichment of the agent from the 287 samples. In the second step 175 urine and serum samples of cattle and horses, collected between 2022 and 2023, were screened for Leptospira-specific immunoglobulins in the Laboratory of the Faculty of Veterinary Medicine of the University of Leipzig, Germany. In detail, ligA and lipL32 genes of Leptospira interrogans serovar Hardjo were cloned into the pQE30 expression vector. Recombinant proteins were evaluated by SDS-PAGE and Western Blot. rLipL32 and rLigA proteins were used to detect anti-Leptospira IgGs in urine and serum samples by Western Blot and to evaluate anti-Leptospira IgG, IgM, IgA by ELISA. Bovine urine samples with low total protein amount were used for cut-off value calculation. In order to increase the reliability of the developed ELISA method, urine samples were pre-adsorbed to eliminate possible E. coli-specific antibodies. ELISA results before and after pre-adsorption were compared. A total of 143 (49.83%) samples were determined positive by 16srRNA PCR. In detail, 44.64%, 48.39% and 87.5% of the cattle, sheep and horse samples were positive for 16srRNA gene, respectively. Furthermore, 26.79% and 25.81% of the cattle and sheep samples were positive for lipL32, respectively. cimA gene was detected in 4.02% of cattle and 16.13% of the sheep samples. lipL32 and cimA could not be detected in horse samples. Only 3 out of 287 cultural analyzed samples were positive for Leptospira shown by 16srRNA PCR. These samples were fresh urine samples. Two of them were PCR positive for the pathogenic Leptospira gene lipL32. Anti-Leptospira immunoglobulins were detected in urine and serum samples of cattle and horses by Western Blot and ELISA. In the bovine urine ELISA with rLipL32 36.71%, 38.28% and 29.68% of the samples were determined positive for IgG, IgM and IgA, respectively. In the ELISA with rLigA, 46.88%, 25.78% and 28.90% were positive for IgG, IgM and IgA, respectively. Pre-adsorption of the urine samples increased the specificity of the ELISA, but did not change significantly the results. Before pre-absorption, 44.53% and 46.09% of the samples were positive in the rLipL32 and rLigA ELISA, respectively. After pre-absorption, 42.97% and 45.31% of the samples were positive in the rLipL32 and rLigA ELISA, respectively. ELISA was positive although there were MAT negative results from blood sera from the same cattle. It can be concluded: I) that the prevalence of leptospirosis has increased in our country and should not be ignored, II) that the developed indirect ELISA method, based on rLipL32 and rLigA, can be used in the analysis of specific IgG, IgA, IgM in urine samples and III) that the most suitable sample for the isolation of Leptospira is fresh urine.
Açıklama
Anahtar Kelimeler
ELISA, İzolasyon, Leptospira, Pre-adsorpsiyon, Western Blot, Isolation
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Gök, A., (2023). Farklı Hayvan Türlerinde Atığa Sebep Olan Leptospirozisin Moleküler Epidemiyolojisi. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimler Enstitüsü, Konya.
