Mehmet Burak ATEŞ, Funda TERZİ, Mustafa Kemal ÇİFTÇİ, Mustafa ORTATATLI, Zeynep ÇELİK

Detection of Botulinum Neurotoxin Serotypes C and D, and Their Eff ects on Expressions of SNAP-25 and Synaptobrevin in Ruminants: An Immunohistochemical Study

In humans and animals, botulism is a disease characterized by generalized and progressive paralysis caused by Clostridium botulinum neurotoxins (BoNT). BoNTs, defined in seven diff erent antigenic types (A to G), proteolyze SNAREs (synaptosomal-associated protein/SNAP-25 and synaptobrevin) responsible for acetylcholine release in peripheral cholinergic neurons, and thus cause fl accid paralysis and death. Currently, mouse experiments are considered the reference method for definitive diagnosis. However, new diagnostic methods that are fast and accurate and would not raise ethical issues need to be developed. Therefore, using antibodies specific to the toxoid forms of BoNTs, the presence of BoNT-C and/or BoNT-D was investigated by immunohistochemical method (IHC) in the study. The tissues of thirty ruminants (twenty cattle, seven sheep, three goats), which had the clinical and pathological findings of botulism and a herd history of the disease, were used as material. BoNTs were detected with IHC in sixteen of the thirty ruminants as three BoNT-C, eleven BoNT-D, and two BoNT C+D. In the mouse experiments, BoNT was isolated in only three cases (two BoNT-D, one BoNT-C). Additionally, being responsible for the clinical findings of botulism, the interaction of BoNTs with SNAP-25 and synaptobrevin was investigated using IHC. It was determined that BoNT-C specifically reduces the expression of SNAP-25, and BoNT-D reduces the expression of synaptobrevin and partially SNAP-25. It was concluded that additional studies may be valuable to investigate the use of IHC in the diagnosis of botulism.

Ruminantlarda Botulinum Nörotoksin Serotip C ve D’nin Saptanması ve Bunların SNAP-25 ve Sinaptobrevin Ekspresyonları Üzerindeki Etkileri: İmmunohistokimyasal Bir Çalışma

Botulizm insan ve hayvanlarda Clostridium botulinum nörotoksinlerinin (BoNT) neden olduğu, generalize ve ilerleyici paraliz ile karakterize bir hastalıktır. Yedi farklı antijenik tipi (A dan G’ye) tanımlanan BoNT periferik kolinerjik nöronlardaki asetilkolin salınımından sorumlu olan SNARE proteinlerini (SNAP-25 ve sinaptobrevin) proteolize ederek fl asid paralize ve ölüme neden olur. Günümüzde fare deneyleri hastalığın kesin teşhisi için referans yöntem olarak kabul edilmektedir. Ancak, hızlı, güvenilir ve etik problemlere yol açmayan yeni teşhis metotlarının geliştirilmesi gerekmektedir. Yapılan çalışma ile botulismus sürü öyküsü, klinik ve patolojik bulguları bulunan 30 adet ruminanta (20 adet sığır, 7 adet koyun ve 3 adet keçi) ait çeşitli dokular BoNT-C ve BoNT-D yönünden immunohistokimyasal (IHC) yöntemle BoNT’un toksoid formuna spesifik antikorlar kullanılarak incelenmiştir. Bu kapsamda 30 ruminantın 16 (3 BoNT-C, 11 BoNT-D ve 2 BoNT C+D)’sında IHC metodu ile BoNT tespit edildi. Fare deneylerinde ise yalnızca 3 vakada BoNT (2; BoNT-D, 1; BoNT-C) izole edildi. Ayrıca, botulizm klinik bulgularından sorumlu olan SNAP-25 ve sinaptobrevinin BoNT ile olan etkileşimi IHC ile araştırılmış ve BoNT-C’nin ruminantlarda spesifik olarak SNAP-25’i, BoNT-D’nin ise spesifik olarak sinaptobrevini kısmi olarak da SNAP-25’i proteolize ettiği saptanmıştır. Botulizm tanısında IHC kullanımını araştırmak için ek çalışmaların yapılmasının değerli olabileceği sonucuna varıldı.

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