Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology

The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.     

Methods for investigating Trichinella infections in domestic and wild animals

Trichinellosis is an important parasitic zoonosis that is caused by the intracellular nematode Trichinella spp.. Infection of humans occurs through consumption of raw (or undercooked) meat containing infectious larvae. In Europe, meat from pork, horse, and wild boar have been identified as most important sources of Trichinella infections in humans. In Switzerland, both the domestic pig and wild boar population are considered free of Trichinella. Conversely, Swiss foxes, lynxs and recently a wolf were found to be infected, the species identified in these animals was always referred to as Trichinella britovi. Although this species rarely infects pork and, compared to Trichinella spiralis, only causes reduced pathogenic effects in humans, the basic presence of Trichinella in Switzerland cannot be neglegted. This fact has gained increasing importance since the responsible authorities in the European Union (EU) are preparing regulations for the official Trichinella-control in meat in order to improve food safety for consumers. These regulations will be implemented as a consequence of the recent association of east European countries with the EU. This new legislation particularly takes into account, that in the past by far most cases of human trichinellosis in the EU were due to consumption of imported east European meat.Within the framework of the bilateral agreements of Switzerland with the EU, the Swiss veterinary public health authorities will have to comply with the foreseen EU regulations. Although diagnostic methods for the direct demonstation of Trichinella in pork meat are already routine practice in several Swiss abattoirs, the implementation of a meat control program for Trichinella for the entire slaughter pig population of the country would lead to an enormous increase in costs for the administration and will require an increased infrastructure in veterinary services. In order to find a reduced testing format for monitoring Trichinella infections in Swiss pork, an infection risk-oriented survey strategy is currently evaluated. In the present article, this minimized survey strategy is discussed regarding its compatibility with the EU regulations laying down rules for the official control of meat for Trichinella.     

Human outbreak of trichinellosis in the Mediterranean island of Sardinia, Italy

Trichinella sp. infection has been documented in both humans and animals in most Mediterranean countries, yet in the past 60 years no infections have been reported on the Mediterranean islands. We describe the first outbreak of Trichinella sp. infection to have been reported on the island of Sardinia. The outbreak occurred in two villages in 2005 and involved 11 persons who had eaten raw sausages made from the same free-ranging sow. All 11 persons developed signs and symptoms of trichinellosis and seroconverted within 48 days of consuming the infected meat. The etiological agent was Trichinella britovi. Sardinia, like all Mediterranean islands, had been considered to be Trichinella-free. The present report, together with a recent report of T. britovi infection in animals on the nearby island of Corsica (France), raises questions as to the validity of the concept of Trichinella-free areas or regions.     

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.     

Trichinae certification in the United States pork industry

Control of Trichinella infection in U.S. pork has traditionally been accomplished by inspection of individual carcasses at slaughter or by post-slaughter processing to inactivate parasites. We propose that an alternative to individual carcass testing or processing can be used when pigs are raised in production systems where risk of exposure to Trichinella spiralis has been mitigated. Declines in prevalence of this parasite in U.S. domestic swine during the last 30 years, coupled with improvements in pork production systems, now allow Trichinella control to be shifted to the farm through implementation of specific pork production practices. Knowledge of risk factors for exposure of swine to T. spiralis was used to develop an objective audit of risk that can be applied to pork production sites. In a pilot study, 461 production site audits were performed by trained veterinary practitioners. The on-farm audit included aspects of farm management, bio-security, feed and feed storage, rodent control programs and general hygiene. Of the 461 production site audits, 450 audits (97.6%) indicated compliance with the required good production practices. These sites are eligible for certification under the U.S. Trichinae Certification Program and will be audited regularly to maintain that status. The described trichinae certification mechanism will establish a process for ensuring the Trichinella safety of swine, and ultimately food products derived from swine, at the production level.     

First isolation of Trichinella britovi from a wild boar (Sus scrofa) in Belgium

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.     

Detection and surveillance for animal trichinellosis in GB

The zoonotic disease trichinellosis is considered one of the re-emerging diseases with surveillance and control methods constantly gaining more importance worldwide. Recent change in European Union (EU) legislation introduces Trichinella-free production, and the possibility of risk-based monitoring for Trichinella in pigs. This has increased the role of wildlife surveillance programmes and their impact on protecting human health as well as highlighted the need for harmonised surveillance protocols and test methods for these infections. A modified digest method, based on the EU reference method for Trichinella testing of pig meat, was used to screen foxes present in Great Britain (England, Scotland and Wales) for trichinellosis. The method was validated using batched pools of 10 g foreleg muscle from up to 20 foxes (maximum amount 200 g). The method gave an average trichinae recovery rate of 71% for spiked samples. Assuming this recovery rate applies to all contaminated samples, then the test sensitivity would be 70% for all tissue samples with 0.1 trichinae per 10 g of foreleg muscle, 99.9% for samples with 1 trichinae per 10 g, and 100% for samples with 2 or more trichinae per 10 g. In two separate studies, conducted between 1999 to 2001 (Smith et al., 2003) and 2003 to 2007, over 3500 wild foxes have been screened for Trichinella with negative results. In the second study reported here, foxes were collected from locations throughout Great Britain using a stratified sampling method based on fox population densities. All work was conducted in compliance with appropriate quality assurance systems, latterly under ISO 9001. Results to date indicate the national prevalence of trichinellosis in foxes is <0.001 based on a 10 g individual sample size, an infection level of 1 larva per gram (l pg) and 95% confidence interval. This, together with no reports of trichinellosis in domesticated pigs, suggests that Britain can be considered a region of negligible risk of trichinellosis.     

Verification of Good Production Practices that Reduce the Risk of Exposure of Pigs to Trichinella

Control of Trichinella infection in pork has traditionally been accomplished by inspection of individual carcasses at slaughter or by post-slaughter processing to inactivate parasites. A decline in the prevalence of this parasite in domestic swine during the last 20 to 30 yr coupled with improvements in pork production systems offer the opportunity to document pork safety at the farm level. We report here on a certification pilot study using an on-farm audit to document good production practices for swine relative to the risk of exposure to trichinae. Based on the results, improvements in the program have been made and further studies will be undertaken prior to launching the voluntary Trichinae Certification Program in the U. S.     

Towards a standardised surveillance for Trichinella in the European Union

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.     

Autochthonous and imported Trichinella isolates in Germany

The study of Trichinella isolates from wildlife in Germany revealed the presence of Trichinella spiralis and Trichinella britovi in wild boars and foxes. T spiralis was detected in meat products imported from Spain, which is one of the two endemic areas of domestic trichinellosis in the European Union: It was also detected in meat from a grizzly bear marketed in Alaska, and Trichinella nativa was detected in a polar bear from the Berlin Zoo. These results stress the importance of examining for Trichinella live animals and meat products imported to Germany from both EU and non-EU countries. Furthermore, carnivores from Arctic regions that are born in the wild and placed in zoos can represent a risk for the introduction of the freeze-resistant species of Trichinella in a new region if, once the animal dies, the carcass is not properly destroyed.     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

New patterns of Trichinella infection

Human and animal trichinellosis should be considered as both an emerging and reemerging disease. The reemergence of the domestic cycle has been due to an increased prevalence of Trichinella spiralis, which has been primarily related to a breakdown of government veterinary services and state farms (e.g., in countries of the former USSR, Bulgaria, Romania), economic problems and war (e.g., in countries of the former Yugoslavia), resulting in a sharp increase in the occurrence of this infection in swine herds in the 1990s, with a prevalence of up to 50% in villages in Byelorussia, Croatia, Latvia, Lithuania, Romania, Russia, Serbia, and the Ukraine, among other countries. The prevalence has also increased following an increase in the number of small farms (Argentina, China, Mexico, etc.) and due to the general belief that trichinellosis was a problem only until the 1960s. The sylvatic cycle has been studied in depth at both the epidemiological and biological level, showing the existence of different etiological agents (Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella nelsoni) in different regions and the existence of "new" transmission patterns. Furthermore, the role of game animals as a source of infection for humans has greatly increased both in developed and developing countries (Bulgaria, Canada, Lithuania, some EU countries, Russia, USA, etc.). The new emerging patterns are related to non-encapsulated species of Trichinella (Trichinella pseudospiralis, Trichinella papuae, Trichinella sp.), infecting a wide spectrum of hosts (humans, mammals including marsupials, birds and crocodiles) and to encapsulated species (T. spiralis, T. britovi, and T. murrelli) infecting herbivores (mainly horses). The existence of non-encapsulated species infecting mammals, birds and crocodiles had probably remained unknown because of the difficulties in detecting larvae in muscle tissues and for the lack of knowledge on the role of birds and crocodiles as a reservoir of Trichinella. On the other hand, it is not known whether horse and crocodile infections existed in the past, and their occurrence has been related to improper human behavior in breeding. The problem of horse-meat trichinellosis is restricted to France and Italy, the only two countries where horse-meat is eaten raw, whereas mutton and beef have been found to be infected with Trichinella sp. only in China.     

Trichinella in horses: a low frequency infection with high human risk

After the initial report in 1976 of a trichinellosis epidemic caused by the consumption of infected horsemeat, 12 other outbreaks have been described in Europe. Since the first serious human outbreak several experiments have confirmed the susceptibility of horses to Trichinella species and the rapid disappearance of specific antibodies in this host that prevents the use of serological methods for routine screening. A review of the distribution of parasite burdens in muscles of naturally or experimentally infected horses indicates that the tongue is the most likely sample to contain detectable numbers of Trichinella larvae in low level infections. Requirements for testing of horsemeat are specified in legislation of the European Union, and other recommendations are published elsewhere. The EEC directives have evolved into very specific requirements which specify the testing of at least 5g of tongue, masseter or diaphragm per horse using a pooled digestion assay. More recently, France has revised the requirement for sample size to 10g for horsemeat originating from countries with high prevalence of Trichinella. To address the continuing outbreaks of human trichinellosis due to infected horsemeat, the development and implementation of a quality assurance system for testing is being considered.     

Control of trichinellosis by inspection and farm management practices

The prevention of human trichinellosis by proper meat inspection is a classic example of successful veterinary public health measures. The microscopic methods which have been used for more than a century to test pigs for trichinae were intended to prevent human disease. However, the value of these relatively insensitive direct detection methods, including trichinoscopy and pooled sample digestion, was debated as soon as more sensitive indirect (serological) methods became available. Two issues related to testing were discussed. First, should public health authorities endeavour to prevent all infections of humans rather than simply prevent the occurrence of disease, and second, would epidemiological surveillance and monitoring of the pig population on farms not provide a better control system to prevent human infection. This latter issue is of particular importance for those countries in the world where human trichinellosis acquired from farmed animals is absent and examination of pigs at the abattoir only results in negative findings. In countries where domestic pig infections are virtually non-existent, monitoring of Trichinella infection in wildlife could also contribute to understanding the infection pressure from nature to livestock. Trichinella-free pig farming is a feasible option for controlling this zoonosis, even in endemic areas. This approach provides an opportunity to combine good veterinary practice, in order to prevent animal diseases, with the prevention of Trichinella infection. All animals with access to the environment, or animals which are fed with potentially Trichinella-infected feed (swill, carcasses) will always constitute a public health threat, and must be inspected individually at slaughter (swine, horses, wild boars). Finally, it is important to recognize that trichinellosis is a world-wide problem that needs continuous public health attention. If no control system exists, for whatever reason, the public should be educated not to consume improperly cooked meat.     

Destruction of Trichinella spiralis spiralis during the preparation of the "dry cured" pork products proscuitto, proscuittini and Genoa salami

Genoa salami, proscuittini and proscuitto were prepared from pork carcasses that were heavily infected experimentally with Trichinella spiralis spiralis. Genoa salami was prepared with salt concentrations of 2.0%, 2.75% and 3.3%. Proscuitto was prepared by two procedures approved by Agriculture Canada. At various times postpreparation, samples of the various cured products were taken and examined by pepsin digestion and rat bioassay for the presence of viable trichinae. Water activity and pH of the cured meat were also determined. Curing of the various products was shown to destroy the Trichinella larvae. Pepsin digestion revealed that larvae progressively became loosely coiled, uncoiled and more subject to digestion (ghost larvae) during the curing process. Rat bioassay revealed the presence of viable trichinae in the proscuitto prepared using a sodium chloride salt mixture at day 34 but not at day 48 postpreparation. All other bioassays carried out on Genoa salami between 13 and 42 days postpreparation, on proscuittini between days 27 and 69 and on proscuitto between days 34 and 69 were negative for viable trichinae. Under the conditions of this study, preparing Genoa salami with salt concentrations as low as 2% did not appear to affect the destruction of Trichinella larvae.     

Epidemiology of trichinellosis in Mexico, Central and South America

Trichinella species are widely distributed throughout the world and are found in a large number of carnivorous animals, humans and incidental hosts. The data presented in this review show that Trichinella infection has been reported in both humans and animals in Mexico, Argentina and Chile since the end of the 19th century, and more recently in Bolivia. This parasitic infection is still a public health problem in countries such as Argentina and Chile. Although efforts have focused on the control and prevention of trichinellosis in these countries, there were still human cases and outbreaks of trichinellosis reported. Diagnosis of infection in animals such as pigs still includes, in many endemic areas, the use of trichinoscopy. In Argentina, however, artificial digestion has been recently introduced in slaughterhouses to detect Trichinella infection in pigs. In some endemic areas in Mexico, the use of serological assays for human trichinellosis and pig infections have resulted in improved detection. Most of the outbreaks of human trichinellosis in Mexico, Argentina and Chile have occurred as a result of the consumption of undercooked pork or pork products from animals raised under poor hygienic conditions and which are clandestinely slaughtered. In several studies, rats, dogs and cats have been found to be infected with Trichinella and may be considered a risk for transmission of the infection to pigs or other animals intended for human consumption. Another potential source of transmission of Trichinella to humans is horsemeat; however, information related to horse trichinellosis in Latin-American countries is scarce. In most cases the etiological agent of human trichinellosis in Central and South America has been reported to be Trichinella spiralis; however, only in a few cases has the parasite species been properly identified. Based on the reports available, it is clear that there is a need to carry out better controlled epidemiological studies to determine the true prevalence of the infection in this region of the world. Also, more sensitive methods of diagnosis and improvements in conditions for pig production as well as better sanitary inspection systems, are needed for controlling and preventing transmission of trichinellosis in these countries.     

National and EU Pathologies and Norms in Mollusc Culture

In the 1990s EEC countries drafted several directives concerning the aquaculture sector, including Directive 91/67/EEC of 28.01.1991 (accepted into Italy by DPR n. 555 on 10.12.1992) and Directive 95/70/EC from 22.12.1995 (accepted into Italy by DPR n. 395 on 20.10.1998). DPR 555 includes among its provisions a list of the diseases that are so serious for production that they must be eradicated from the EEC territory (Annex A, List I), as well as a list of other diseases that also affect production (Annex A, List II).Annex A, List II of DPR 555, includes two mollusc diseases, namely Bonamiosis and Marteiliosis for flat oysters (Ostrea edulis). DPR 395 includes among its provisions that farm owners register their farm with the National Health Service and possess a loading and unloading register of the material introduced from or destined for another farm for reintroduction.When the National Health Service finds a case of unusual death in the wild or in farms, it will apply a sanitary monitoring programme to ascertain or exclude the presence of the diseases included in Annex A to 91/67/EEC or Annex D to 95/70/EC as the cause of death. On February 5th 2003 the EU Commission modified Annex D (2003/83/EC).     

Cattle transport regulations in the European Union

The essential items of the relevant EU legislation are described and their effects are discussed. A brief survey of the four most important legal regulations in the EU relevant for cattle transport is given from the point of view of administration. These regulations are: The Directive 91/628/EEC 1991 (protection of animals during transport), Council Regulation (EC) No 1255/97 (criteria for staging points and route plan), Council Regulation (EC) No 411/98 (additional standards for road vehicles for long distance transport), Commission Regulation (EC) No 615/98 (export refund regulations). Some critical points of the objectives are pointed out.     

Reliability evaluation of sampling plan fixed by Council Directive 91/68/EEC for the maintenance of officially brucellosis-free flock status

The European Union (EU) strategy with respect to sheep and goat brucellosis aims to eradicate the infection and achieve officially brucellosis-free (OBF) status in all EU holdings and territories. Council Directive 91/68/EEC of 28 January 1991 states that to maintain OBF status of ovine or caprine holdings located outside an OBF territory, only a representative number of animals need to be tested annually. However, depending on the number of animals in a holding, this testing method risks non-detection of the infection, thereby reducing the efficacy of the brucellosis control plan. The recommended sampling procedure has a low sensitivity for detecting infection in medium-sized flocks; furthermore, the risk of not detecting re-infection in OBF flocks, particularly in territories that have not yet gained OBF status, is also not acceptable. Moreover, in large-sized flocks, the Directive sampling procedure entails taking an excessive number of samples, which can be very expensive. The authors evaluated, by using statistical analyses and a simulation model based on field data, the possible consequences of the current EU strategy. It is suggested that the sampling criteria for the maintenance of OBF status in the EU should be modified and that a statistically based sampling method should be applied instead of the fixed percentage method that is currently in use.     

National and EU pathologies and norms in mollusc culture

In the 1990s EEC countries drafted several directives concerning the aquaculture sector, including Directive 91/67/EEC of 28.01.1991 (accepted into Italy by DPR n. 555 on 10.12.1992) and Directive 95/70/EC from 22.12.1995 (accepted into Italy by DPR n. 395 on 20.10.1998). DPR 555 includes among its provisions a list of the diseases that are so serious for production that they must be eradicated from the EEC territory (Annex A, List I), as well as a list of other diseases that also affect production (Annex A, List II). Annex A, List II of DPR 555, includes two mollusc diseases, namely Bonamiosis and Marteiliosis for flat oysters (Ostrea edulis). DPR 395 includes among its provisions that farm owners register their farm with the National Health Service and possess a loading and unloading register of the material introduced from or destined for another farm for reintroduction. When the National Health Service finds a case of unusual death in the wild or in farms, it will apply a sanitary monitoring programme to ascertain or exclude the presence of the diseases included in Annex A to 91/67/EEC or Annex D to 95/70/EC as the cause of death. On February 5th 2003 the EU Commission modified Annex D (2003/83/EC).