Emmanuel Vanopdenbosch

Biological risks associated with consumption of reptile products.

The consumption of a wide variety of species of reptiles caught from the wild has been an important source of protein for humans world-wide for millennia. Terrapins, snakes, lizards, crocodiles and iguanas are now farmed and the consumption and trade of their meat and other edible products have recently increased in some areas of the world. Biological risks associated with the consumption of products from both farmed and wild reptile meat and eggs include infections caused by bacteria (Salmonella spp., Vibrio spp.), parasites (Spirometra, Trichinella, Gnathostoma, pentastomids), as well as intoxications by biotoxins. For crocodiles, Salmonella spp. constitute a significant public health risk due to the high intestinal carrier rate which is reflected in an equally high contamination rate in their fresh and frozen meat. There is a lack of information about the presence of Salmonella spp. in meat from other edible reptilians, though captive reptiles used as pets (lizards or turtles) are frequently carriers of these bacteria in Europe. Parasitic protozoa in reptiles represent a negligible risk for public health compared to parasitic metazoans, of which trichinellosis, pentastomiasis, gnathostomiasis and sparganosis can be acquired through consumption of contaminated crocodile, monitor lizard, turtle and snake meat, respectively. Other reptiles, although found to harbour the above parasites, have not been implicated with their transmission to humans. Freezing treatment inactivates Spirometra and Trichinella in crocodile meat, while the effectiveness of freezing of other reptilian meat is unknown. Biotoxins that accumulate in the flesh of sea turtles may cause chelonitoxism, a type of food poisoning with a high mortality rate in humans. Infections by fungi, including yeasts, and viruses widely occur in reptiles but have not been linked to a human health risk through the contamination of their meat. Currently there are no indications that natural transmissible spongiform encephalopathies (TSEs) occur in reptilians. The feeding of farmed reptiles with non-processed and recycled animal products is likely to increase the occurrence of biological hazards in reptile meat. Application of GHP, GMP and HACCP procedures, respectively at farm and slaughterhouse level, is crucial for controlling the hazards.

Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy.

ABSTRACT Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.

Natural case of bovine herpesvirus 1 meningoencephalitis in an adult cow

Mim) and thinner (width 0-25 to 0-3 urm) than the cells of the other Brachyspira species, and have four to six fewer axial filaments (Stanton and others 1997, Trivett-Moore and others 1998). Stanton and others (1997) found it difficult to distinguish between the cells ofB hyodysenteriae, Brachyspira intermedia and Brachyspira murdochii, based on cell dimensions, cell morphology and numbers of axial filaments. The histological changes of Brachyspira species infection in the cow in the present study were broadly similar to those described in swine dysentery and porcine colonic spirochaetosis, with the exception of the previously described end-on attachment of B pilosicoli to the surface epithelium (Taylor and others 1980, Thomson and others 1996). Immunohistochemically, the organisms were stained positively by polyclonal antisera against B hyodysenteriae and B pilosicoli, but because of the low specificity of those antisera, immunohistochemical examination can only be used to identify Brachyspira species. In swine dysentery, the lesions are restricted to the colon, caecum, and rectum, but they are most constant and severe in the spiral colon; however, the caecum and the proximal colonic mucosa were severely affected with the spirochaetes in this case. These differences in the lesions and organism predilections among studies were partly attributed to the differences in the organism species and host species. Taking these results and the pathology of swine dysentery, this dairy cow case is the counterpart of swine dysentery. Since attempts to isolate the spirochaetes in this case failed, the exact identity of the spirochaetes is still unknown. Further studies with different media and culture conditions will be necessary to try and isolate spirochaetes from cattle with dysentery.

Scientific Opinion on a quantitative estimate of the public health impact of setting a new target for the reduction of Salmonella in laying hens

Public health risks of Salmonella infection in laying hens (Gallus gallus) can be associated with exposure through four different pathways: internally contaminated table eggs, externally contaminated table eggs, egg products and meat from spent hens. In relation to eggs, Salmonella Enteritidis is by far the serovar most frequently associated with human illness, and exposure through eggs that are internally contaminated with this serovar has a higher public health significance than exposure to externally contaminated eggs. A mathematical model, using reported field data from two EU Member States (MSs), suggests a linear relationship between the investigated scenarios of flock prevalence for Salmonella Enteritidis and the number of contaminated eggs that would be laid. However, the absolute public health impact of the assessed flock prevalence scenarios is highly uncertain due to lack of data on the number of contaminated eggs produced by infected flocks and on the true number of egg-related human salmonellosis cases. It is suggested that public health benefits, similar to those obtained reaching lower Salmonella flock prevalences, may be achieved by implementing controls based on more sensitive sampling protocols. Diversion of eggs from flocks that are tested positive in the EU Salmonella control programme to the production of egg products subjected to heat treatment may lead to increased health risks as heat treatment of egg products should not be considered an absolute barrier to Salmonella contamination. Fresh meat from spent laying hens might carry a higher prevalence of Salmonella than meat from broiler flocks, in particular if sourced from Salmonella-positive flocks. The quantification of under-ascertainment and underreporting of human salmonellosis cases, improving knowledge on within-flock dynamics of Salmonella and harvesting data on production of Salmonella contaminated eggs under field conditions would contribute to improving the accuracy of future quantitative estimates.

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and CR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.

Trends in age at detection in cases of bovine spongiform encephalopathy in Belgium: an indicator of the epidemic curve

There were 118 cases of bovine spongiform encephalopathy (bse) in Belgium before January 1, 2004. Trends in their age at detection were analysed and attempts were made to use this parameter as a predictor of the current status of the bse epidemic in the country. The following variables were considered: date of birth, breed, date of detection, mode of detection, and the number and age of animals slaughtered and rendered each month. Age at detection as a function of date of birth was a very poor epidemiological indicator. It was concluded that the increasing age of bse cases when they were detected was due to the depletion of cases, as a result of there being no new infections, and that it is a reliable indicator of a decrease in the epidemic curve in Belgium. By means of a simulation it is shown how age distribution at the time of detection closely follows the epidemic curve and data from Great Britain are used to illustrate the point.

Population-Level Retrospective Study of Neurologically Expressed Disorders in Ruminants before the Onset of Bovine Spongiform Encephalopathy (BSE) in Belgium, a BSE Risk III Country

ABSTRACT A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.

Mycobacterium bovis meningitis in a cow with clinical signs of BSE.

Lic, Dcpartmenit of Biocontrol, K. Walravens, PhD, J. Godfroid, D)MNi, Department of Bacteriology and Imimunology, Veterinary and Agrochemical Research Centre (NAR) Ukkel, Groeselenberg 99, B1 180 Brussels, Belgium C. Saegerman, D)XVM, Ceintral Veterinary Services, T( III, Simon Bolivarlaan, B1000 Brussels, Belgium M. Thelissen, DMNI, Lic, Veteriinary Services of Limburg, B-3500 Hasselt, Belgiuum Mycobacterium bovis belongs to the Mycobacterium tuberculosis complex consisting of a highly related group of acid-fast bacilli, and is the main cause of tuberculosis (TB) in cattle and other wild or domestic mammals (Cousins 2001). The prevalence ofM bovis in small ruminants and pigs is low and is apparently proportional to that in major hosts such as cattle (Gutierrez and others 1998, Serraino and others 1999). M bovis has been sporadically isolated in cats and dogs and in wild mammals in captivity. Wildlife reservoirs ofM bovis have been described in badgers in the UK (Delahay and others 2001) and in the brushtailed possum in New Zealand (Coleman and Cooke 2001). M bovis infections have also been described in buffaloes in southern Africa and in bison in North America, as well as in a number of species of freeranging deer (Morris and others 1994, De Lisle and others 2002). Human beings are also susceptible, usually acquiring the infection as a result of the ingestion of infected milk products (Murray 1990, Jones and others 1997). The organism is maintained and spread in cattle principally by aerosol, but calves can be infected in utero or by the ingestion of milk (Bolin and others 1997, Jones and others 1997). Animals infected with M bovis are recognised as a zoonotic threat, and the disease is notifiable in many countries (Directives EE(, 64/432, EU 92/117). The annual herd incidence and prevalence in Belgium in 2001 were 0-044 per cent and 0-024 per cent, respectively. This short communication describes an incident of M bovis meningitis in a cow which showed clinical signs consistent with bovine spongiform encephalopathy (BSE). In January 2002, a red-white heifer, aged 16 months and originating from an 85-cow dairy herd, was presented with a history of head tilting and hypersalivation, prominent yawning and snoring (powerful expirations through the nose), followed by the development of an enlarged neck. The veterinarian treated the animal with antibiotics but there was no clinical improvement. The animal developed progressive decubitus and it was finally decided to euthanase the heifer. Subsequently, as a precaution, it was included in the routine protocol for ruminants which are suspected of having a transmissible spongiform encephalopathy (Vanopdenbosch and others 1998). According to the protocol, no postmortem examination could be performed. On removal of the brain, a prominent greyish-white discoloration of the meninges was noticed. This was mainly observed on the cerebellum and all around the brainstem, on the ventral, dorsal and lateral sides. After excluding rabies as a routine precaution, using direct immunofluorescence and isolation on cultures of neuroblastoma cells (Vanopdenbosch and others 1998), samples of the brainstem, the cerebrum and cerebellum were taken for further histological examination (Vanopdenbosch and others 1998). Large samples of these three major brain parts were fixed in a 4 per cent phosphate-buffered formaldehyde solution, processed routinely, embedded in paraffin, and sectioned at 5 pm thickness. Sections were primarily stained with haematoxylin and eosin. The rest of the brain was frozen at -200C. FIG 1: (a) Meninges showing multiple sites of necrosis and multinucleated Langhans giant cells (arrowheads). Haematoxylin and eosin. x 160. (b) Vascular lesions in the meninges showing (peri)vascular infiltration of lymphocytes and plasmacytes and necrosis occasionally associated with the formation of intravascular thrombi (arrows). Haematoxylin and eosin. x 640

Current status of scrapie

Despite being controlled in many developed countries, scrapie remains endemic in many parts of the world. Economic losses result from mortality and culling of small ruminants and from market restrictions. Moreover, it is difficult to develop all-inclusive guidelines that could establish a scrapie-free status for a country. Unfortunately, the global picture remains incomplete because in many countries confusion still remains regarding the clinical picture of scrapie and information is not available owing to the absence of adequate epidemiosurveillance networks. Currently, the predominant theory is that PrP Sc is the infectious agent where host genetic factors play a central role. The precise transmission routes of scrapie and their relative contributions to the overall transmission intensity remain poorly documented and the physiopathology is not fully understood. However, it is evident that the purchase of female sheep from scrapie flocks, sharing pastures with scrapie flocks, sharing breeding rams and genetic host susceptibility are the main risk factors for the spread of the disease. A better understanding of the epidemiology of scrapie would greatly aid the development and evaluation of control and eradication strategies that were mainly based on selective depopulation of infected animals and genetically susceptible and/or related animals and also on the biosecurity and the use of selective genetic breeding programmes in healthy flocks. Some numbers of a new transmissible spongiform encephalopathy (TSE) form in small ruminants (atypical scrapie) have meanwhile been identified by TSE rapid testing using an assay, which also recognizes comparatively less proteinase K-resistant PrP Sc . Uncertainties remain regarding the pathogenesis of this new TSE form, as well as regarding their potential transmissibility within the affected species and to other species. Thus far, no borine spongiform encephalopathy (BSE) cases have been confirmed in sheep under natural conditions (a report of vertical transmission after experimental infection merits attention), but two historical cases of BSE in goats born in the 1990s have been identified. Currently BSE must also be considered in the differential diagnosis of scrapie. The development of prevention and control programmes should be assisted by new scientific findings.

Evaluation of the Application for new alternative biodiesel production process for rendered fat of Cat 1 (BDI‐RepCat process, AT)

Abstract A new alternative method for the production of biodiesel from rendered fat of all categories of animal by‐products was assessed. The process was compared to the approved biodiesel production process described in Chapter IV Section 2 D of Annex IV of Commission Regulation (EU) 142/2011. Tallow derived from Category 1 material is treated according to Method 1 from the same Regulation (133°C, 20 min, 3 bar) and subsequently mixed with 15% methanol, heated to reaction temperature (220°C) in several heat exchangers and transferred into the continuous conversion reactor by means of a high pressure pump (80 bar) for 30 min. In the conversion phase, there is an exposure to methanol in the absence of alkaline or acidic conditions. The impact of this procedure on the thermostability of transmissible spongiform encephalopathy (TSE) has not been assessed in the literature. After the reaction, the biodiesel/glycerol mixture is distilled under vacuum at a minimum temperature of 150°C and a maximum pressure of 10 mbar, which is equivalent to the distillation step in the approved biodiesel production process, for which a 3 log10 reduction factor in PrP27–30 was obtained. Therefore, a similar level of TSE infectivity reduction could be expected for that phase of the method. A previous EFSA Opinion established that a reduction of 6 log10 in TSE infectivity should be achieved by any proposed alternative method in order to be equivalent to the approved processing method. This level of reduction has not been shown with experimental trials run under conditions equivalent to the ones described for the RepCat process. It was not possible to conclude whether or not the level of TSE infectivity reduction in the RepCat process is at least of 6 log10. Therefore, it was also not possible to conclude about the equivalence with the approved biodiesel production process.