Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429):Trypanosoma evansi infections (including Surra)

Abstract Trypanosoma evansi infections (including Surra) have been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of T. evansi infections (including Surra) to be listed, Article 9 for the categorisation of T. evansi infections (including Surra) according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to T. evansi infections (including Surra). The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, it is inconclusive whether T. evansi infections (including Surra) can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL because there was no full consensus on the criterion 5 A(v). Consequently, the assessment on compliance of T. evansi infections (including Surra) with the criteria as in sections 4 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (d) and (e) of Article 9(1) is also inconclusive, as well as which animal species can be considered to be listed for T. evansi infections (including Surra) according to Article 8(3) of the AHL

Farklı toxocara vitulorum antijenlerinin elde edilmesi ve enfeksiyona spesifik antikorların saptanması

Bu çalışmada, Türkiye’de şimdiye kadar dışkı, kolostrum/süt ve otopsi bakıları ile bildirilen ve hala bazı yörelerde buzağılarda sorun olabilen T. vitulorum’unserodiagnozu amaçlandı. Toxocara vitulorum’la doğal enfekte buzağı serumlarında,hazırlanan biri larva (Ex) diğeri olgun (Pe) antijenle humoral immun yanıt araştırıldı.Araştırmanın laboratuar çalışmalarında için gerekli materyallerin (dıskı,kan, parazit) sağlanması için önce saha çalışmalarına yoğunlaşıldı ve T. vitulorumile enfekte hayvanlar arandı. Değişik 22 ilden; yaşları 0-6 aylık; halk elinden, özel ve resmi kurumlardan; yerli, kültür ırkı ve melez; her iki cinsiyetten 2393 buzağıya ait dışkı Fulleborn doymuş tuzlu su flotasyon yöntemi ile kontrol edildi. Yirmi sekiz örnekte (% 1,17) T. vitulorum yumurtalarına rastlandı. Ankara, Düzce ve Erzurum illerine ait dışkılarda enfeksiyona rastlandı, resmi kurumlara ait dışkıların hiçbirinde yumurta görülmedi. McMaster tekniği ile gram dışkı yumurta sayısı en fazla10 250 olarak belirlendi.Toxocara vitulorum larva (Ex) antijeni hazırlanmasında; dışkıdan ve olgun parazitlerden elde edilen yumurtalar oda sıcaklığında gelişmeye bırakıldı. Larva gelişimi 17-40 günde tamamlandı. Yumurta kabuğu soyularak, dısarı çıkması sağlanan larvalar 448,4 (452,6-516,6) Tm boyda 20,1 (14,7-24,6) Tm ende ölçüldü.Toxocara vitulorum olgun (Pe) antijeni hazırlanmasında; kendiliğinden veya ilaç verilerek düşürülen erkek ve dişi parazitler kullanılarak, perienterik sıvı çekildi.Elde edilen larvalardan (Ex), perienterik sıvıdan (Pe) antijenleri seçilen yönteme uygun olarak hazırlandı. Spektrofotometre ile (Ex) antijeni için protein konsantrasyonu 7,5 mg/ml, (Pe) antijen için 20,56 mg/ml olarak belirlendi.SDS-PAGE’le elektroforetik separasyonda (Ex) antijeninde moleküler ağırlıkları 14-150 kDa, (Pe) antijeninde ise 12-141 kDa arasında değişen proteinbantları elde edildi.Toxocara vitulorum (Ex) antijenlerinin negatif serumlarla (4 saha, 3 fötal)immunoblotting analizinde hiçbir örnekle reaktif bant saptanmazken, pozitif 6serumda 14, 35, 56, 63, 68 ve 94 kDa molekül ağırlığındaki protein bantlarının immunoreaktif oldukları tespit edildi. 14 kDa’luk bant bazı örneklerde daha belirgin izlendi. (Pe) antijen ile yapılan immunoblotting çalışmasında negatif serumların (2 saha, 4 fötal) söz konusu antijenlere reaktif olmadıkları, pozitif 6serumun ise tüm örneklerde 96 ve 110 kDa’luk 2 proteine benzer yoğunlukta reaktif oldukları tespit edildi.AbstractToxocara vitulorum causes serious problems among calves in many parts of Turkey.All previous diagnostic approaches for T. vitulorum infections performed based onfaeces, colostrum / milk and post-mortem examinations. The aim of this study is toprepare two kinds of antigens, larva extract (Ex) and perienteric (Pe) forserodiagnosis, besides the advantages of clinical and epidemiological aspects of thedisease in its diagnosis.This study was initiated by an intensive field work that involved the collectionof materials needed for laboratory examinations (faeces, blood and parasites).Faecal samples were collected from a total of 2393 calves of both sexes aged 0-6months, settled in 22 provinces at public/private and state farms of differentbreeds. Then the faecal samples examined in the laboratory by Fulleborn FlotationTechnique using saturated sodium chloride solution. Twenty eight calves werefound positive with T. vitulorum (1,17%) by the presence of the eggs in their faeces.The infected calves were belonged to public/private farms at Ankara, Düzce andErzurum provinces, while no infection was found in calves at the State farms.McMaster technique was performed to determine the parasite egg count per gram offaeces and the maximum (epg) was found as 10250.Toxocara vitulorum eggs were recovered from both infected faeces and themature parasites for the purpose of larval (Ex) antigen preparation. The eggs wereincubated at room temperature when the larval deveopment was completed within17-40 days. The egg shells decorticated and the length and width of the obtainedlarvae was measured as 448,4 (452,6-516,6)Tm and 20,1 (14,7-24,6)Tmrespectively.Toxocara vitulorum perienteric fluid was recovered from both male and femaleparasites that obtained by either the spontaneous explusion of the parasites or theparasites collected after adminstration of drug.Proper procedures used for preparation of (Ex) antigen from the recoveredlarvae and (Pe) antigen from the obtained perienteric fluid. Protein concentration ofboth antigens was measured by spectrophometer where it estimated as 7,5 mg/mlfor (Ex) and 20,56 mg/ml for (Pe) antigen.Different protein bands of variable molecular weights which was estimated as14-150 kDa for (Ex) and 12-141 kDa for (Pe) antigen recovered by SDS-PAGEelectrophoretic separation.The negatif serum samples (4 field, 3 fetal) did not show any reaction bandswith (Ex) antigen in the immunoblotting analysis, while the 6 positive serumsamples showed protein bands within the range of 14, 35, 56, 63, 68 and 94 kDamolecular weights. 14kDa moleculer weight band was more prominent in somesamples.Negative serum samples ( 2 field, 4 fetal) did not react with (Pe) antigen in theimmunoblotting analysis, while all positive 6 serum samples showed proteinbands to 96 and 110 kDa molecular weights with same reactivity