ΓΕΝΙΚΗ Δ/ΝΣΗ ΚΤΗΝΙΑΤΡΙΚΗΣ
Δ/ΝΣΗ ΥΓΕΙΑΣ ΤΩΝ ΖΩΩΝ
Τμήμα Λοιμωδών & Παρασιτικών Νοσημάτων 

Kτηνιατρικές Δραστηριότητες
Ειδικά Προγράμματα Επιτήρησης & Ελέγχου Ασθενειών

HELLENIC REPUBLIC
MINISTRY OF AGRICULTURE
DIR.GENERAL OF VETERINARY SERVICES
ANIMAL HEALTH DIRECTORATE
DEPT. OF INFECTIOUS DISEASES,
EPIDEMIOLOGY & DOCUMENTATION

Address : 2 Acharnon Str.
               101 76 Athens, Greece
Telephone : 0030-1-88 35 420 / 88 36 420
Fax : 0030-1-82 29 188 / 88 36 040
Telex : 22 17 38 YDAG.GR
E-mai: vetserv@ath.forthnet.gr

INTRODUCTION

The purpose of this report  is  to provide an overview  of  the  procedures, modalities  and results of epidemio-surveillance for Transmissible Spongiform Encephalopathies (TSEs) in Greece in 2000, in compliance with Commission Decision  2000/5/EC, and in particular article 31 thereof.

For reasons of clarity and continuity this report is divided into two Parts, namely :

• Part  A, providing  background  information on the  procedures and modalities of       epidemio-surveillance for TSEs, which were not  necessarily  introduced in 2000 but were in place during the reporting period, and
• Part  B, presenting the specific results of surveillance for TSEs obtained in 2000 and discussing some timely and relevant points thereof.

For data of previous years - since 1986 - the reader may browse in the Web Site  of  the Department of Infectious Diseases which is readily accessible  at  the  electronic address :

http://www.minagric.gr/greek/2.3.1html

PART A : GENERAL FRAMEWORK OF SURVEILLANCE

For the purposes of this report, TSEs shall mean (a) BSE in bovines and (b)  Scrapie in sheep and goats. Progressive disorders compatible with a TSE-syndrome in  otheranimal species are also investigated and reported, where applicable.

The framework of surveillance for TSEs in Greece is based on three main pillars, i.e :

1.      Passive (Random) Surveillance

This is based on the fact  that  both  BSE and  Scrapie are compulsorily  notifiable in Greece. Specifically :

• BSE has been compulsorily notifiable, in a legally binding manner, since 1992 by Presidential Decree 133/92 transposing Commission Decision 92/450/EC.
• Scrapie has been compulsorily notifiable, in a legally binding manner, since 1995 by Presidential Decree 138/95 transposing Council Directive 91/68/EC.
  However, Scrapie  has  been  sought  for, reported and  acted upon in an ad-hoc manner since 1986, when the first case ever was reported in Greece.

As a consequence, veterinarians (official or private practitioners),farmers, associated professionals and the agricultural community at large  are  required  to  report  to  the competent authorities all suspect cases of TSEs.

Disease  awareness  among  professionals  and  the  industry  has  been consistently promoted since  the early ‘90s  through a  wide  variety  of  suitable  means, including publications in daily press and professional  journals, videos, lectures, documentaries and/or talk shows on TV, both at national and local networks.

2.      Active (Targeted) Surveillance

Since 1997 a national programme for active  surveillance  and  control  of  TSEs  has been in  place in Greece. This  programme  is  continuously  reviewed  in  the light  of  scientific advances and epidemiological findings and was last amended by Ministerial Decision No.331734 / 04.02.2000.

The current programme  for surveillance and control of TSEs takes on board relevant requirements and guidelines laid  down  in  Commission  Decision  98/272/EC and  in  Chapters 3.2.13 and 3.3.8 and Appendix 4.5.1 of the OIE Animal Health Code.

The programme was approved and co-financed by the Community for  the year 2000 by Commission Decision 2000/5/EC, and in particular article 31 thereof.

Briefly, the programme addresses and specifies issues such as :

• Objectives of surveillance.
• Involved Services and their job description in the framework of surveillance.
• Nomination of National Reference Laboratories.
• Definitions of suspect cases, confirmed cases, sub-populations at risk etc.
• Appropriate clinical, laboratory and epidemiological investigations.
• Measures on suspicion and confirmation.
  * NOTE : During  the reporting period a selective stamping out policy was applied,          depending on the within-herd prevalence and the incidence propagation. 
• Additional measures, supplementary to the objectives of surveillance.
• Compensation arrangements and funding of relevant activities.

3.      Scientific Advisory Committee on TSEs

By Ministerial Decision No.307169 / 09.04.1997, a  Scientific  Advisory  Committee on TSEs was established in Greece comprising of prominent  scientists  and researchers from the University and senior veterinary offices from the Ministry of Agriculture.

The Scientific Advisory Committee meets regularly or on an extraordinary basis and is mandated to :

• Review and assess the latest scientific evidence available on TSEs.
• Devise and supervise epidemiological investigations inside Greece.
• Carry out relevant animal and public health risk assessment studies.
• Advice on appropriate surveillance, control and eradication measures.
• Transfer and implement in Greece appropriate diagnostic tests and protocols.
• Identify research priorities and plan and/or conduct suitable research projects.
• Liaison with other national competent authorities (i.e Ministry of Health).

During the reporting period the Scientific Advisory Committee met on five  occasions  for the following purposes :

• In February, to  review and deliver an  opinion  on  the  draft  proposal  amending Commission Decision 98/272/EC,  
• In April, to make suitable proposals for adapting  the  Greek Programme intended for implementation from the year 2001 in  conformity  with  Commission  Decision 2000/374/EC,
• Twice in November, to consider the modalities for  implementation of Commission Decisions 2000/764/EC and 2000/766/EC,
• In December, to deliver an opinion on proper strategies for  the implementation of Commission Regulation (EC) Nr.2777/2000/EC and comment on the new  revised  Programme for Surveillance & Control of TSEs for the year 2001. 

On these occasions, the Scientific Advisory Committee monitored closely the current epidemiological situation of  TSEs in Greece, discussed  the  latest  results  available and advised on specific relevant issues, such as  disinfection procedures, restocking of depopulated premises, disposal of carcasses etc.    

PART B : MODALITIES & RESULTS OF SURVEILLANCE

1.      Modalities of surveillance

In the field, surveillance  for TSEs is organized on a step-wise procedure on the basis of  the  following  decision tree for  the investigation, and confirmation or ruling out, of suspect cases :

      Step 1 :  Clinical suspicion and differential diagnosis of TSE, including response to
                    treatment. 
                    Approximately 80% of disorders involving the CNS and/or the locomotive
                    systems or inducing a change of normal behavioral patterns giving  raise
                    to suspicion of TSEs may be confidently  attributed to causes other  than
                    TSEs on the basis of clinical symptoms, history of the flock and response
                    to experimental treatment. 
                    Common disorders which can be determined clinically are, in sheep  and
                    goats, enterotoxaimia, Coenurus  Cerebralis and mange and, in bovines, 
                    ketosis.

                    * NOTE : Accumulative results of clinical findings with a possible bearing on 
                                  TSEs obtained  during  2000 under a Step 1 procedure are
                                  summarized in Table 1.  

   Step 2 :     Differential laboratory diagnosis,obtained in the first instance,of diseases  
                    other than TSEs. 
                    Approximately 15% of disorders involving the CNS and/or  the locomotive
                    systems may be definitively attributed to diseases or disorders other  than 
                    TSEs by common laboratory routinely tests  performed  in any  one  of 17
                    Regional Veterinary Labs operating in Greece.
                    Common diseases  which may be determined by routine laboratory  tests
                    are, in sheep and goats, listeriosis, encephalitis, brain tumors and  Maedi-
                    Visna and, in bovines, encephalitis and malignant catarrhal fever.

                    * NOTE : Accumulative results of routine laboratory tests  performed  in  
                                  2000 under a Step 2 procedure are summarized in Table 2.

      Step 3 :  Specialized laboratory testing, in the second instance, of suspect cases
                    where TSEs could not be ruled out on the basis of  clinical diagnosis or 
                    routine laboratory tests.
                    Approximately 5% of disorders involving the CNS and/or the locomotive
                    systems can not be diagnosed clinically or through a  routine laboratory
                    test and, consequently, are referred to the National  Reference Lab  for
                    thorough and specialized  tests leading to confirmation or  ruling out  of
                    TSE.
                    Samples are forwarded to the National Reference Lab for  TSEs  either
                    by one of the Regional Veterinary Labs, in case  of  failure  to  reach  a
                    definitive diagnosis through a Step 2 procedure, or directly by the  field
                    Services, when the clinical  symptoms, the  epidemiological  conditions
                    or the history of the individual animal/flock indicate an increased risk of
                    a true TSE case.

                    * NOTE : Detailed results of laboratory tests performed in 2000  under  
                                  a  Step 3  procedure  are  summarized  in Table 3 and  their
                                  origin is indicated Maps 1 and 2, depending on the species.

2.      Results of surveillance – Presentation

Following the path-way  described in  paragraph 1 above, the  combined  results  of passive and active surveillance for TSEs  obtained in Greece in 2000 are as follows.  

2.1  Clinical surveillance 

Results of the most common clinical investigations with a potential bearing on TSEs carried out  in the reporting period under  a  Step 1  procedure  are  summarized  in Table 1 below. Cases were evenly distributed throughout the country.

Table 1 : Results of clinical investigations of TSE-suspect cases obtained in 2000.

Source   :  Regular returns submitted by Regional  Veterinary Services

Comment on Table 1: Due to the nature of the disorders, figures provided in Table 1 
                                must  be considered as  indicative  and  are  probably  largely
                               underestimated because of under-reporting.  

2.2   Routine laboratory tests

Results of tests performed during the reporting period under a  Step 2  procedure are summarized in Table 2. Samples originated in the entire country

Table 2 : Accumulative results of routine laboratory tests obtained in 2000.

Source   : Combined regular returns submitted by Regional Veterinary Services & Laboratories

Comment on Table 2 : Figures  of  certain disorders, and  in  particular  metabolic
                                 disorders and poisonings,are probably underestimated due
                                 to certain under-detecting and/or -reporting.

2.3  Specialized laboratory tests

Results of tests performed during the reporting period under a Step 3 procedure are summarized in Table 3, and the origin of samples is indicated in Maps 1 and 2.

Table 3 : Detailed results of specialized laboratory tests for TSEs

Source   : Special reports submitted by the National TSE Reference Laboratory 

Comments on Table 3

1. The reference test used for confirmation of TSEs was histo-pathology which was augmented, in most cases, by other supplementary tests  such  as  western  blot   for the purpose of epidemiological investigations (see paragraph 4).
2. Flocks from which samples were submitted break down as follows :

•  20 sheep flocks
•    2 goat flocks
•  12 mixed flocks (sheep & goats)

3. All Scrapie-positive samples were obtained from sheep,although the disease may occur naturally in goats as well.
4. As a rule, increased number of samples were tested form Scrapie-affected flocks.
   This practice was due to  :

   -   Either, propagation of the disease is some flocks, with several
       suspect  cases appearing over a period and requiring repeated
       tests,
   -   Or, testing of all animals in the cohort of the index case.

   This practice may  cause a distortion  of  the  estimated  ratio  of  3 :1 as regards tests performed under a Step 2 and Step 3 procedure.

3.      Results of surveillance – Discussion

On the basis of combined results presented in Tables 1, 2 and 3, the following points of epidemiological significance emerge :

3.1  The nation-wide incidence of confirmed cases of BSE and Scrapie in 2000 is : 

• For BSE      :  0,0 cases
• For Scrapie :  6,7 cases / million of susceptible animals (over 12 months)

3.2  The average annual incidence for 2000 of neurological disorders in which TSEs  could not be ruled out on clinical grounds is :

• For bovines            :  0,10 % (in cattle over 24 months of age)
• For sheep & goats :  0,04 % (in sheep/goats over 12 months of age)

3.3    The main epidemiological characteristics of Scrapie cases detected in 2000 are :

• 10 out of 11 flocks affected by Scrapie were detected for the first time in 2000 in the framework of the programme.
•  In 8 out of 11 flocks  affected by Scrapie  within-flock  prevalence  was > 2 %.    over a period of several months.
  This was the established threshold for applying a stamping out policy.      
• Accordingly, 1.523 sheep/goats were killed and destroyed  in  the 8 “heavily” affected flocks.
• The remaining 3 affected flocks have been placed under  official restrictions and are subject to close monitoring to establish  the  incidence  propagation rate and determine appropriate eradication measures.
• The age of the individually affected animals ranged between 2 and 4 years.
• All affected animals were native-born in old and well established flocks.     
• The spatial distribution of affected flocks, as indicated in Map 2, suggests a geographical concentration and persistence, manly in Northern Greece. 
  The explanation may be that long-term backwards tracing of affected flocks points to organized efforts for genetic improvement, undertaken in  the  late ‘70s and early ‘80s in Northern Greece, by means  of  imported  pure-breed animals.
  Indeed, affected flocks are invariably genetically improved through pure- or cross-breeding with Suffolk, Frisian, Frisarta, Chios (for sheep) and Zaanen (for goats – observed in previous years) breeds.  
• Genetic related susceptibility has been confirmed  in  all  positive  cases  by PrP Condon analysis.   

3.4   Despite increased disease awareness, no case  of TSE  has been detected  in         animal species other than sheep and goats.

4.  Supplementary & Related Actions

With a view to further enhance epidemio-surveillance for, and eventually eradication of, TSEs  in Greece  the following  supplementary  actions  have  continued or been launched in 2000.  

4.1    Characterization of Scrapie strains to determine whether they  are  actually  due  to  BSE agent or not. For this purpose two strains isolated in Kilkis in 1998 from  contiguous flocks, one of sheep and one of goats, have been sent  to Germany and one strain isolated in Thessaloniki  in 1998 has  been  sent  to  France   for nucleotide sequence analysis following bio-assay in mice. 
Results are not available to this date.

4.2     Field research aiming to conduct a genetic screening of Chios pure-breed rams  reared in Genetic  Improvement and A.I Centres supervised  by  the Ministry  of  Agriculture as stock for  genetic  improvement (semen donors)  with  a  view  to identify and remove from reproduction genetically susceptible animals.

In the framework of this  project  genotyping  of 110 sheep of  the Chios  breed has been carried out  for  PrP  polymorphisms  in  codons 136, 154 and 171 by means of Denaturing Gradient Gel Electrophoresis (DGGE).

Preliminary  results suggest a high genetic susceptibility of  screened  animals,  while publication of final results is pending.

4.3    A number of  relevant  research activities and  projects  are  in  progress  using material from Scrapie-infected animals. Such projects include, in particular :

1. Case-control studies with the purpose of defining Scrapie "susceptible" and "resistant" PrP genotypes in Greek sheep and goat breeds.
   The methodology involves collection of samples from clinically affected and healthy animals in same flock(s) and testing of these samples for PrP geno-type, by histo-pathology and western blot.
   Final results are not available to this date.
2. Association of goat PrP polymorphisms with Scrapie. Provisional results of sequencing the PrP gene reveal polymorphisms in condons 42 (silent),138 (silent), 143, 154 and 240. Definitive results are not available to this date.
   The DGGE method, using sheep and goat markers, is also being validated.

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This concludes the Annual Report on epidemio-surveillance of TSEs in Greece in 2000