Bovine spongiform encephalopathy in Italian herds

Bovine spongiform encephalopathy in Italian herds
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  SHORT COMMUNICATIONS- Bovine spongiformencephalopathy in Italian herds M. CARAMELLI,   ACUTIs, E. BOZZETTA, C. CASALONE, C. GAGNA, G. Ru BOVINE spongiformencephalopathy (BSE) is a fatal disease of cattle, which is associated with the accumulation of an abnormal isoform of the prion protein PrPres. Strong evidence indicatesthat the agent of BSE is the cause of the new variant of Creutzfeldt-Jakob disease in human beings (Bruce and oth- ers 1997, Hill and others1997). The concern for public health and the pitfalls of passive surveillanceled to the implemen- tation of EU-wide active surveillance based on rapid tests, at the beginning of 2001. Previously, in Italy, only a couple of cases of the disease had beenfound in 1994 in animals imported from the UK  Guarda and others 1994). The aim of this short communication is to providepreliminary infor- mation on thedescriptive epidemiology and neuropathology of BSE in Italy. In January 2001, a network of 18 state laboratories (within the Istituti Zooprofilattici Sperimentali) was set up to carry out the rapid immunoblotting test for thedetection of PrPres (Prionics Check; Prionics) (Schaller and others1999) on all animals over 30 months of age. All the animals tested had been slaughtered or came from fallen stock. A database of all the Italian testing is maintained by the NationalReference Centre (CEA). Positive cases were officially confirmed by the CEA. The brainstemofeachanimal subjected to the rapid test was collected and stored at 40C at the state laboratories, until the rapid test result was known. If the test was positive, the brainstem sample was submitted to the CEA, where the obex and pons at the caudal cerebellar peduncles were formalin fixed for histological and immunohistochemical (IHC)analy- sis. A fresh sample taken from the same area as that used for the rapid test was evaluated by Western blot  Wb) analysis. Paraffin-embedded sections were partlystained withhaematoxylin and eosin and partly immunohistochemically stained by the avidin/biotin peroxidase method (Vectastain ABC kit; Vector Lab), applying two enhancing pretreat- ments: 98 per cent formic acid for 20 minutes and hydrated autoclaving at 121°C for 30 minutes. The primary antibody was the polyclonal rabbit antiserum P7/7 (1:500)raised against purified PrP 27-30 of hamster brain infected with the 263K scrapie strain (Pocchiari and others 1994). For Wb, PrP was detected with 6H4 antibody (Prionics) and a chemiluminescent substrate  CDP Star; Bio-Rad). Finally, a retrospectiveinvestigation of theanimal s clinical presentation and epidemiological information was per- formed. During the first eight months of 2001, 227,360 samples were examinedand 26 cases of BSE w r detected (1 14 cases of BSE per 10,000 animals tested) (Table 1 . The mean age of the animals was six years  range four to 13 years). Twenty-four of the 26 infected herds (92-3 per cent) were located insix northern regions which contain around 70 per cent of Italian dairyfarms; a statistically significant excess of cases in this area was evident (24 observed cases v  8 expected cases with a P<0.05using a Poisson distrib- ution). Only three animals were born before theintroduction of the feed ban in 1994. None of the cases detected had been indicated as suspect: nine hadbeen classified in the risk cat- egories as defined by the EU Decision 2001/8/EC (three fallenstock, three emergency slaughtered cows and three animals with non-specific clinical signs at antemortem examination). The remaining animals were slaughtered routinely, but from theretrospectiveinvestigation only three cases were healthy, whereas reductionofmilk yield,loss of body condition and a posterior paresis were reported in 13 cases. In one case, overt neurological signs including reluctance to pass through a m Cumulative Date of Sampling number birth date Target group Breed Country Region of srcin HE Veterinary Record  2003) 153,711-712 M. Caramelli, DVM, PhD, P. Acutis, DVM, E. Bozzetta, DVM, C. Casalone, DVM, C. Gagna, DVM, PhD, G. Ru, DVM, PhD, CEA   Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta, Via Bologna 148, Torino, Italy 1/2001 2/2001 3/2001 4/20015/20016/2001 7/2001 8/20019/2001 10/2001 11/2001 12/200113/200114/2001 15/2001 16/2001 17/2001 18/2001 19/2001 20/2001 21/2001 22/200123/200124/200125/200126/2001 1/9/941/1/961/8/951/2/971/5/941/11/96 1/12/94 1/11/941/9/94 1/3/95 1/10/95 1/8/95 3/6/9410/10/9625/7/95 23/12/96 6/11/8717/3/96 25/10/96 1/10/9512/9/95 10/5/97 31/5/961/1/97 9/12/96 1/9/96 10/1/01 3/2/0115/2/01 3/2/01 22/2/01 10/3/01 15/3/01 17/3/0120/3/01 20/3/01 23/3/01 4/4/01 26/4/018/5/0115/5/01 27/6/01 3/7/01 5/7/01 11/7/01 13/7/01 23/7/01 2/8/01 18/8/01 22/8/01 28/8/01 28/8/01 Healthy slaughteredanimals Holstein Fallen stock Holstein Healthyslaughteredanimals Holstein Emergency slaughtered animals Simmental lik Fallen stock Brown SwissHealthyslaughteredanimals Brown Swiss Emergency slaughteredanimals Holstein Fallen stockHolstein Healthy slaughteredanimals Holstein Healthy slaughteredanimals Holstein Healthy slaughteredanimals Holstein Healthy slaughtered animals Brown Swiss Healthy slaughtered animals Brown Swiss Healthy slaughtered animals Brown SwissHealthy slaughtered animals Holstein Animals withc dinical signs Brown Swiss antemortem (not BSE suspect) Emergency slaughteredanimals HerensAnimals with dinical signs Holstein antemortem  not BSE suspect) Healthy slaughtered animals Brown Swiss Headhy slaughtered animals Holstein Healthy slaughtered animals Holstein Healthy slaughteredanimals Brown Sw iss Animals wi dinical signsHolstein antemortem (not BSE suspect)Healthyslaughtered animals Brown SwissAnimals with dinical signs Holstein antemortem (not BSE suspect)Healthy slaughtered animals Holstein Lombardia Italy Lombardia Italy Lombardia Italy e Friuli Italy Puglia Italy Lombardia Germany Marche Italy Lombardia Italy Lombardia Italy Lombardia Italy Emilia Romagna Italy Veneto Italy Veneto Italy Piemonte Italy Lombardia Italy Lombardia Italy Valle d Aosta Italy Lombardia Italy Emilia Romagna Italy Lombardia Italy Lombardia Italy Lombardia Italy Lombardia Italy Veneto Italy Emilia Romagna Italy Lombardia Italy The VeterinaryRecord, December 6, 2003 Analysis IHC Wb   t Pos Pos Pos Pos PosPosPos PosPos Pos Pos PosPos Pos Pos Pos Pos PosPosPos PosPos Pos Pos Pos Pos HE Haematoxylin and eosin, IHC Immunohistochemistry, Wb Western blot, Pos Positive, +, ++,   Indicate degrees of staining  l 711  group.bmj.comon July 15, 2014 - Published by veterinaryrecord.bmj.comDownloaded from   SHORT COMMUNICATIONS FIG 1: PrP immunostaining in the medulla at the level of the obex incattle withbovine spongifonn encephalopathy. Linear tracts and granular deposition. P7/7 antibody. x 200 FIG 2: PrP immunostaining in the medulla at the level of the obex in cattle with bovine spongifomn encephalopathy. PrP fibrillar accumultion in the neuropil. P7/7 antibody. x 400 doorway, aggressiveness and apprehension had been observed. Histologically, all except two cases showed conspicuous spongiosis in the neuropil in thesolitary tract nucleus and in the nucleus of the spinal tract of the vagus nerve, whereas mild spongiosis wasobserved in thedorsal motor nucleus of the vagus nerve. Vacuolisation ofneuronal perikarya was predominantly seen in the vestibular nuclear complex, in reticular formation and, to a lesserextent,in thedorsal nucleus of the vagus nerve. The PrP deposition showed three distinct major patternsthat appeared widespread through- out the neuropil and did not appear to be related to theseverity of spongiosis.Fig 1 shows fine or coarse, granulardeposition and beaded linear tracts, while Fig 2 shows fibrillar accumulation. The analysis of the glycoform patterns revealed that the PrPres was constant in all BSE samples. The Italian BSE cases resembled thosedescribed in other European countries regarding both the neuropathology (Wells and others1992, Orge and others 2000, Tegtmeier and others 2001) and the epidemiology (Wilesmith and others 1988). The main difference between cases concerned the reported clinical presentation. It is not clear if unlike in othercountries,the Italiancases were characterised mostly by mild and unspecific signs or if there was a problem in the recog- nising and reporting of BSE-consistent signs. With the excep- tion of two cases, the severe pathological patterns observed suggested that animals were in a late stage of the disease. However, the relationship between the neuropathology and the clinical progression of the disease needs further investigation. ACKNOWLEDGEMENTS The authors wish to thank L. Capucci, F. Mutinelli, A. Petrella and S. Barocci for supplying pathological material. The anti- body P7/7 was a gift from M. Pocchiari, Istituto Superiore di Sanita, Roma. The authors arealso grateful to 0. Bugiani and G. Giaccone, at the Istituto Neurologico Carlo Besta, Milan, for their invaluable collaboration. This work was supported in part by the Italian Ministry of Health, Dipartimento AlimentazioneNutrizione SanitA Veterinaria Pubblica and Consiglio Nazionale delle Richerche, Comitato Scienze Agrarie, Progetto Strategico BSE. BRUCE, M. E., WILL, R. G., IRONSIDE, J. W., MCCONNELL, I., DRUM-MOND, I., SUTTIE, A., MCCARDLE, A., CHREE, A., HOPE, J., BIRKETT, C., COUSENS, S., FRASER, H.   BOSTOCK, C. J (1997) Transmission to mice indicatesthat  new variant CiD is caused by the BSE agent. Nature 389, 498-501 GUARDA, F., CASTIGLIONE, F., AGRIMI, U., CARDONE, F., CARACAPPA, S.   POCCHIARI, M. (1994) Encefalopatia Spongiforme Bovina (BSE) in Italia. Progresso Veterinario 21, 3-7 HILL, A. F., DESBRULAIS, M., JOINER, S., SIDLE, K C., GOWLAND, I., COLLINGE, J DOEY, L. J LANTOS, P. (1997) The same prion strain causes vCJD and BSE. Nature 389,448-450 ORGE, L., SIMAS, J P., FERNANDES, A. C., RAMOS, M.   GALO, A. (2000) Similarity of the lesion profile of BSE in Portugese cattle to that described in British cattle. Veterinary Record 147, 486-488 POCCHIARI, M., XI, Y. G., INGROSSO, L., LADOGANA, A., CARDONE, F., MASULLO, C., RIGHETTO, Z., BIGON, E., DI MARTINO, A.   CALLE- GARO, L. (1994) Immunodiagnosis ofbovine spongiform encephalopathy. Livestock Production Sciences38, 41-46 SCHALLER, O., FATZER, R., STACK, M., CLARK, J COOLEY, W., BIFFIGER, K., EGLI, S., DOHERR, M., VANDEVELDE, M., HEIM, D., OESCH, B.   MOSER, M   (1999) Validation of a Western immunoblotting procedure for bovine PrPsc detection and its use as rapid surveillance method for the diag- nosis ofbovine spongiform encephalopathy (BsE). Acta Neuropathologica 98, 437-443 TEGTMEIER, C., AGERHOLM, J S., BILLE-HANSEN, V., SCHAAP, P. K.   RYDER, S. (2001) First confirmed native case of spongiform encephalopathy in Denmark. Veterinary Record 148, 51-52 WELLS, G. A. H., HAWKINS, S. A. C., HADLOW, W. J SPENCER Y. I (1992) The discovery of bovine spongiform encephalopathy and observation on the vacuolarchanges. In Prion Diseases of Humans and Animals. Eds S. B. Prusiner, J. Collinge, J. Powell, B. Anderton. Chichester, Ellis-Horwood. pp 256-274 WILESMITH, J. W., WELLS, G. A. H., CRANWELL, M. P.   RYAN, J. B. M. (1988) Bovine spongiform encephalopathy:epidemiological studies. Veterinary Record 123, 638-644 Notices and divisional events DIVISIONS of the BVA are entitled to a free notice in The Veterinary Record for each meeting that they organise. Notices should containthe date,time, venue and town of the meeting, details of the subject, any speakers and sponsors and the address and telephone number of the person from whom details can be obtained. They should be addressed to: Kathryn Clark, The Veterinary Record, 7 Mansfield Street, LondonWIG 9NQ, fax 0207637 0620. The VeteWinary Record, December 6, 200312  group.bmj.comon July 15, 2014 - Published by veterinaryrecord.bmj.comDownloaded from   doi: 10.1136/vr.153.23.711  2003 153: 711-712 Veterinary Record   M. Caramelli, P. Acutis, E. Bozzetta, et al.  Italian herdsBovine spongiform encephalopathy in Updated information and services can be found at: These include:  References Article cited in: serviceEmail alerting the box at the top right corner of the online article.Receive free email alerts when new articles cite this article. Sign up in Notes To request permissions go to: To order reprints go to: To subscribe to BMJ go to:  group.bmj.comon July 15, 2014 - Published by veterinaryrecord.bmj.comDownloaded from 
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