Interlaboratory reproducibility of a test method following 4-field test methodology to evaluate the susceptibility of Clostridium difficile spores.

BACKGROUND: Sporicidal surface disinfection is recommended to control transmission of Clostridium difficile in healthcare facilities. EN 17126 provides a method to determine the sporicidal activity in suspension and has been approved as a European standard. In addition, a sporicidal surface test has been proposed. AIM: To determine the interlaboratory reproducibility of a test method for evaluating the susceptibility of a C. difficile spore preparation to a biocidal formulation following the 4-field test (EN 16615 methodology). METHODS: Nine laboratories participated. C. difficile NCTC 13366 spores were used. Glutaraldehyde (1% and 6%; 15 min) and peracetic acid (PAA; 0.01% and 0.04%; 15 min) were used to determine the spores' susceptibility in suspension in triplicate. FINDINGS: One-percent glutaraldehyde revealed a mean decimal log10 reduction of 1.03 with variable results in the nine laboratories (0.37-1.49) and a reproducibility of 0.38. The effect of 6% glutaraldehyde was stronger (mean: 2.05; range: 0.96-4.29; reproducibility: 0.86). PAA revealed similar results. An exemplary biocidal formulation based on 5% PAA was used at 0.5% (non-effective concentration) and 4% (effective concentration) to determine the sporicidal efficacy (4-field test) under clean conditions in triplicate with a contact time of 15 min. When used at 0.5% it demonstrated an overall log10 reduction of 2.68 (range: 2.35-3.57) and at 4% of 4.61 (range: 3.82-5.71). The residual contamination on the three primarily uncontaminated test fields was <50 cfu/25 cm2 in one out of nine laboratories (0.5%) and in seven out of nine laboratories (4%). CONCLUSION: The interlaboratory reproducibility seems to be robust.

The trade network in the dairy industry and its implication for the spread of contamination.

In case of an outbreak of a foodborne disease, administrative decisions in the context of crisis management are only efficient if they follow standard practices and are specifically adapted to the outbreak situation in a timely manner. These goals are hard to achieve. The complexity of national and global trade structures obscures a clear view of trade flows and, consequently, it is often impossible to unravel complex trade links quickly. Furthermore, increasing public concerns about possible health hazards caused by global trade put additional pressure on decision makers. The aim of this paper was to unveil the specific trade structures of the German milk supply chain, to highlight how these structures could affect the spatial spread of a hypothetical contaminant, and to quantify the risk of the contaminant reaching the consumer. To achieve this goal, the vertical and horizontal trade links between milk producers, dairies, and consumers were taken into account. The horizontal flow of milk between dairies (inter-dairy trade), which is intended to compensate a temporary over- or undersupply of milk, is of special importance in this respect. We hypothesized that the extent of inter-dairy trade would significantly influence the spatial spread of contaminated milk and the contamination risk. This hypothesis was tested using a computer simulation model that predicts the hypothetical spread of a contaminant via trade of milk. The model parameters were estimated using trade data collected in 2004 and 2010. The results of our study indicate that inter-dairy trade significantly influenced the contamination risk. Compared with a scenario with no inter-dairy trade, the risk that contaminated milk will reach the consumer was up to 4 times higher, even with moderate inter-dairy trade. The contamination risk depended on the extent of inter-dairy trade in a nonlinear way and reached its maximum asymptotically when inter-dairy trade increased. The contamination risk exhibited considerable spatial variation, which could be utilized to implement more accurate food control interventions in times of crisis caused by a foodborne disease.

A long-term serological survey on Aujeszky's disease virus infections in wild boar in East Germany.

Between 1985 and 2008, a total of 102,387 wild boar sera originating from Eastern Germany covering an area of 108 589 km2 were tested for the presence of Aujeszky's disease virus (ADV)-specific antibodies. From 1985 until 1991 and from 1992 until 2008, wild boar sera were exclusively investigated using either conventional seroneutralization assays (n=39 621) or commercial gB and full antigen ELISAs (n=62,766), respectively. Spatial-temporal analysis revealed an increasing ADV seroprevalence from 0·4% to 15·9%, on average, during the 24-year observation period that went along with a continuous spread of the infection in a western direction. During 2006 and 2008, 18% of the 66 affected districts had ADV seroprevalences >30%. There was a significant correlation between ADV seroprevalence and the hunting index of population density (HIPD) of wild boar in the entire study area, although this did not hold true for some regions. Seroprevalences did not differ between sexes but were age-dependent. East Germany has been officially free of Aujeszky's disease (pseudorabies) in domestic pigs since 1985. Although a risk for domestic pigs cannot be completely ruled out, experience has shown that ADV in domestic pigs could be eliminated although the virus was present in the wild boar population. Despite increasing ADV seroprevalence in the East German wild boar population no spillover infections from wild boar to domestic pigs have been reported. To further trace ADV infections in the wild boar population in Germany, a nationwide serological monitoring programme should be implemented.

Trade communities and their spatial patterns in the German pork production network.

The German trade network of pig holdings and related enterprises for the time period of 01 June 2006-31 December 2008 was analyzed. Available data comprised of the entire German trade information with about 121,287 pig premises and their links in the pork production chain (breeders to slaughter houses). During the study period, 330,000 trade connections between premises were recorded. Communities which are large scale structures comprising of premises which are in close trade contact with each other were detected by modularity maximization. Almost 97% of the pig holdings could be assigned to one of nine major communities. Contacts between communities are rare. Trade communities do not only form spatial clusters, but were also associated with specific regions within the territory of Germany. Communities are to some extent separated by 'trade borders', which may impede disease spread. Interestingly, the trade borders often failed to match with administrative borders of the German federal states. This finding is important, because it illustrates the need for new strategies of cross-border disease management strategies.

Scenario-analysis evaluating semergency strategies after rabies re-introduction.

Now that the elimination of rabies in Western Europe is nearly complete, thanks to the oral mass vaccination of foxes (ORV) which took place over the last 25 years, it is necessary to prepare for emergency situations due to the re-introduction of rabies from still infected areas. Such emergency strategies should aim at minimizing the risk of falling back to large-scale vaccination, in a cost efficient manner. An approved spatially-explicit simulation model of spread and control of rabies was adapted to the new problem of re-introduction of rabies into free areas. The logic of the model and options for local emergency vaccination (for example ring-vaccination vs. compact area treatment or heavily concentrated vs. thin extended control areas) were determined. Based on systematic simulation experiments the performance of strategic options was assessed. Key issues such as public health risk (i.e. number of rabies cases), failure risk (i.e. disease breakout from the control area), and budgetary risk (i.e. duration of the emergency program) were simultaneously considered. The results obtained reveal efficiency relations that contradict a priori derived management suggestions.

The financial challenge of keeping a large region rabies-free--the EU example.

Following the implementation of oral rabies vaccination of foxes (ORV) in Western Europe, a continuous decrease in rabies incidence was reported, and eventually rabies was eliminated. Once fox rabies is eliminated in a given area, re-infection from neighbouring infected countries is a permanent threat. As a result, countries need to maintain a vaccination belt along common borders until rabies is also eliminated in sufficiently large border regions of neighbouring infected countries. In a theoretical approach EU member states were taken as a prime example, assuming that they were rabies-free but that neighbouring countries were still infected. Using GIS, a 50 km deep vaccination belt beyond the front of the rabies endemic zone was installed in countries bordering those regions. The annual cost for the prevention of re-infection of the EU territory was calculated considering current EU recommendations (vaccination twice per year, aerial and complementary hand distribution, bait density of 30 baits per km2). Minimum and maximum prices for commercial available oral rabies vaccine baits, aircraft and rabies surveillance were considered for the calculation of costs. The total vaccination area which needed to be established was about 251,000 km2. Using mainly fixed-wing aircraft, the annual cost for ORV including rabies surveillance varied between a minimum of 10 and a maximum of 16M Euro, depending on the cost of vaccine bait. If helicopters were used exclusively, the maximum cost increased to about 32M Euro. Depending on the length of the border to infected regions, countries will have to pay up to 25% of the total cost. Countries which need to install a vaccination belt will never have a rabies-free status because of the likely occurrence of rabies cases in border zones.

What is the future of wildlife rabies control in Europe?

Over the last fifteen years or so, classical rabies in terrestrial wildlife has been eliminated from large areas of Western Europe. Over the next few years, terrestrial rabies is likely to occur only east of a line from the Baltic Sea to the Black Sea; the overall aim is to eliminate terrestrial rabies from the whole European Union. Elimination of rabies from the less rich countries of Eastern Europe, and the protection of Europe against a resurgence of rabies in the longer term requires modifications to existing OIE and WHO strategies. Here we discuss the options available to eliminate rabies in wildlife while taking account of financial cost, and how to maintain a 'cordon sanitaire' along the eastern boundary of the EU in order to protect the rabies-free areas from rabies incursion. Minimising financial costs at the national level is obviously essential, considering the competing priorities for development and health. This could be achieved either by increasing external funding (for example by the EU) and/or by changing the currently agreed vaccination strategy to reduce costs; any such change must not substantially reduce the chances of rabies elimination. A cordon sanitaire might be placed outside the economic area of the EU, to protect the whole of the EU, or it might be placed within the easternmost countries to ensure logistical consistency of vaccination. Policy must also anticipate an emergency due to rabies breaking out in a previously freed region. Strategic planning may be complicated by the increasing range and abundance of the raccoon dog, an introduced species that is increasingly important as a host for fox rabies. It is argued here that models help to evaluate altemative strategies, exploring options for optimising costs by minimising bait density and frequency or by reducing the vaccination area.

A random grid based molecular epidemiological study on EBLV isolates from Germany.

Germany has reported one of the highest levels of EBLV cases in bats in Europe. So far, all isolates originating from Germany have been identified as EBLV-1, using monoclonal antibodies, and a preliminary epidemiological study has indicated that there is a distinct geographical distribution of EBLV-1 in Germany. To further investigate the spatial and temporal distribution of EBLV-1 variants in Germany and their impact on molecular epidemiology, isolates were selected using a random grid sampling procedure based on GIS. Agrid layer 30 km long over the entire area of Germany was applied to 120 geo-referenced isolates and one isolate of each grid cell containing EBLV isolates was randomly chosen. Once selected, the nucleoprotein (N) plus parts of the phosphoprotein (P) gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on European EBLVs, showing a high sequence homology between German EBLV-1 isolates. Almost identical sequence homologies within certain geographical regions indicate genomic stability during the transmission cycle of EBLV-1, with little geographic spread or intermixing. Interestingly, a 6 bp insertion as well as a single nucleotide insertion, detected in the N-P intergenic region, has been found in EBLV-1 isolates from Germany.

Risk factors for BSE-infections in Lower Saxony, Germany.

In order to generate data on the cattle population and farm management in the state of Lower Saxony, Germany, a basic demographic survey was conducted. Afterwards these BSE-free farms as a reference population were compared with the population on BSE farms to identify risk factors for BSE infections. A variety of risk factors for BSE were reviewed, including the import of cattle from the United Kingdom, commercial foodstuff, dairy farming, herd size and cross-contamination with foodstuff for other farm animals. For the basic demographic survey of the reference, a questionnaire was mailed to a representative sample of cattle farms in Lower Saxony where BSE cattle had not occurred before the sample was taken. Distribution of risk factors within this reference population (n=731) and the BSE population (n=49) were compared following the concept of indirect standardisation in stratified populations. The size of farms was used as the stratification variable, with three strata. Under the same rate as in the reference population, the portion of Red Holstein cattle breed was four fold higher than in the BSE population (SER=4.03; p=0.0003). Milk replacer was fed 1.41 times more often on BSE farms (p=0.0478). However, the use of concentrated foodstuff for pigs (SER=0.21) was significantly less frequent in the BSE population than expected (p=0.0001), whereas the husbandry of sheep, goats or game animals seemed to increase the risk of BSE 2.85 times (p=0.0413). There were no significant differences between the two populations concerning the purchase of cattle (p=0.1514) and the use of concentrated feed for calves during the 1990s (p=0.6212). This is an epidemiological indication of increased susceptibility of Red Holstein cattle to BSE. However, this study did not confirm the assumption that the use of commercial foodstuff other than milk replacer or the purchase of cattle increases the risk of BSE infection. It nevertheless remains likely that commercial foodstuffs such as concentrated feed for calves were risk factors in Germany as well.

Epidemiology of bat rabies in Germany.

Rabies in European bats was first reported in Germany in 1954. In concordance with Denmark and the Netherlands, Germany has reported one of the highest numbers (n = 187) of European bat lyssavirus (EBLV)-positive cases in bats in Europe so far (1954-2005). A combined descriptive epidemiological and phylogenetic analysis on bat rabies and prevailing EBLVs is presented, comprising the past 50 years. So far, only the two lineages of EBLV-1 (genotype 5), a and b, have been detected. Although only 50% of the rabies-positive bats have been identified by species, the Serotine bat (Eptesicus serotinus) is the bat species most frequently infected. Single rabies cases have also been detected in a further five indigenous bat species. There is proven evidence for a substantial bias in the frequency of bat rabies cases in the north of Germany, with an endemic cluster in the northwesternmost low-lying plain areas adjacent to the Netherlands and Denmark. Improvements to bat rabies surveillance and research are discussed.

Epidemiological analysis of setbacks in oral vaccination in the final stage of fox rabies elimination in densely populated areas in Germany.

Despite a long history of oral vaccination of foxes (OVF) against rabies, in a few restricted areas of Germany rabies is still endemic, posing a continuous risk of re-introduction of rabies into adjacent, rabies-free areas. The endemic area is characterized by a high density population. It is hypothesized that the degree of urbanization in the area under consideration influences the number of rabies cases via the mode of bait distribution. In urban areas vaccine baits are distributed by hand, whereas in non-urban areas baits can be distributed aerially with the help of fixed-winged aircraft. Statistical analysis of the effect of the mode of bait distribution upon the number of rabies cases shows a significant influence. In areas where baits are distributed by hand the number of rabies cases is significantly higher than the expected number. This finding forces managers to reassess the procedure of bait distribution by hand in urban areas, taking into account the ecologically and biologically different dynamics of urban fox populations. If the oral vaccination of foxes in urban areas can be refined, rabies eradication in Germany is expected to succeed in due course.

Cross reactive antigenicity in orally vaccinated foxes and raccoon dogs against European Bat Lyssavirus type 1 and 2.

For the first time, the effectiveness of oral rabies vaccines against European Bat Lyssaviruses Type 1 (EBLV-1) and Type 2 (EBLV-2) by means of cross-neutralization assays was investigated. Sera from orally vaccinated red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) with the Street Alabama Dufferin (SAD) B19 and SAD P5/88 live-modified vaccine viruses were used to study the cross reactive antigenicity against CVS-11 (genotype 1), EBLV-1 (genotype 5) and EBLV-2 (genotype 6). For comparison, similar crossneutralization assays with sera from EBLV-1 and EBLV-2 infected ferrets (Mustela putorius furo) and/or foxes were conducted. Sera from animals vaccinated with the two oral rabies vaccines were reactive against CVS-11 (homologous virus), EBLV-1 and EBLV-2 (heterologous virus). There was a positive relationship among the virus neutralising antibody titres (VNA); high VNA titres against CVS-11 also resulted in high VNA titres against each EBLV, whereas in general, the VNA-titres obtained with homologous virus were statistically higher than those with the heterologous virus except for SAD P5/88 vaccinated raccoon dogs. No significant difference was found between EBLV-1 and EBLV-2 VNA titres. A similar trend was observed when the results of the cross-neutralization data of the foxes and ferrets inoculated i.m. with EBLV-1 and/or EBLV-2 was analysed. Based on the similarity of the EBLV-VNA titres obtained in our study questions were raised on whether the genetic distance of genotype 5 & 6 within phylogroup 1 really does reflect their antigenetic characteristics or whether this is a feature of attenuated live vaccine viruses. This broad cross protection, however, demonstrated that the representatives of attenuated SAD strains of oral rabies vaccines currently used in Germany are most likely able to protect the reservoir species, red fox and raccoon dog, against EBLV-1 and EBLV-2 infection.

Potential risk factors associated with bovine spongiform encephalopathy in cattle from Schleswig-Holstein, Germany.

Since the first detection of bovine spongiform encephalopathy (BSE) in homebred cattle in Germany on 26 November 2000, 382 cases have been confirmed until 30 September 2005. Thirty-two of these cases were reported from the Federal State of Schleswig-Holstein (SH). There are hypotheses on the routes of infection for German cattle, but only few efforts have been made to assess potential risk factors by epidemiological studies. The purpose of this study was to identify potential risk factors at the farm level for the occurrence of BSE in cattle in SH. By applying the method of indirect standardization, the prevalence of various structural and management parameters of BSE-affected farms in SH was compared with the prevalence of these parameters in a standard population of cattle farms from SH. The data describing the standard population were obtained by a cross-sectional study performed in SH in 2003. Data of the BSE case population were available from the central German BSE case database. A possible association of the occurrence of BSE with the feeding of milk replacers to calves was observed. There was a clear indication that the occurrence of BSE was associated with the presence of pigs and/or poultry on the farm. This finding suggests that cross-contamination of feed or cross-exposition may have occurred in SH. The results obtained using the indirect standardization analysis were validated by multiple logistic regression. This study indicates that the feedborne path has been the principal route of transmission for the BSE agent in SH.

Investigations of the potential risk factors associated with cases of bovine spongiform encephalopathy in Bavaria, Germany.

In order to identify the management and feeding practices that might have contributed to the occurrence of bovine spongiform encephalopathy (BSE) in Bavaria, Germany, information from 110 dairy farms on which a case of BSE had been reported was compared with information derived from a questionnaire sent to approximately 10,000 Bavarian farms on which no case of BSE had been reported up to February 2003. Representative information was obtained from 4006 dairy farms. The results indicated that in comparison with these control farms a higher proportion of the BSE farms had also kept pigs or poultry, although the difference was not significant, and that a significantly higher proportion of the BSE farms had fed proprietary concentrates and/or milk replacers to their calves.

Fox rabies in Germany - an update.

In comparison with conventional methods of wildlife rabies control, oral rabies vaccination of foxes (ORV) is without doubt the most (cost-) effective method in wildlife rabies control. As a result of ORV, several European countries have become rabies-free. Although rabies had been eliminated from much of Germany, there still exists a residual rabies focus in the border triangle of Hesse, Baden-Wurttemberg and Rhineland Palatinate. Corrective actions have been initiated to eliminate this last remaining rabies hotspot in Germany.

Long-term studies on maternal immunity for Aujeszky's disease and classical swine fever in wild boar piglets.

The aim of the studies was to fathom the duration and the role of maternal immunity for Aujeszky's disease (AD) and classical swine fever (CSF) in wild boar offspring. In one experiment, two wild boar sows were infected with a low pathogenic pseudorabies virus (PRV) in 1999. A total of 51 offspring was born between 1999 and 2002 and was monitored for PRV maternal antibodies. In a second experiment, the maternal immunity for CSF was analysed. Therefore, a sow was orally vaccinated against CSF using vaccine baits containing the live-attenuated C-strain vaccine. The vaccination took place in January 1999. The sow gave birth to four piglets in 2001 and to two piglets in 2002. With respect to maternal immunity for AD, some piglets reacted positive in the ELISA up to 27-week post-partum while in the neutralization test antibodies were detected up to 15-week post-partum. The calculated half-life of neutralizing antibodies was 21 days. Regarding CSF, the neutralization titres of maternal antibodies dropped continuously reaching values of < or =10 ND50 20-week post-partum. After the 12th week post-partum, most of the sera reacted negative in the ELISA. However, after the third month, low levels of neutralization titres were still detectable. The results are discussed with respect to the epidemiology and control of both diseases in wild boar populations.

[Proposal for changing the spot checking examination regarding the control of the maintenance of freedom from Aujeszky's disease (AD)]. / Vorschlag zur Anderung der Stichprobenuntersuchung bezüglich der Kontrolle zur Aufrechterhaltung der Freiheit von der Aujeszkyschen Krankheit (AK).

In Germany the last outbreak of Aujeszky's disease (AD) in a holding of domestic pigs had been officially confirmed in the year 2000, and, since February 2003 Germany is officially confirmed free of AD. Since this time the AD free status is monitored according to chapter II of the annex of the regulation for the protection against Aujeszky's disease. As an alternative monitoring protocol two variants of a two step random sampling scheme is proposed which reduces the sample size from a complete investigation of all domestic pig holdings to a sample of pig fattening and pig breeding farms. Within the random selected farms, pigs are sampled as usual according to chapter II of the annex of the regulation for the protection against Aujeszky's disease. In this paper, the measures to be taken in case of a positive AD result are discussed: In case of conformation of an AD suspicion it is proposed to establish a restriction area. In addition measures based on regionalisation are proposed in order to maintain the AD free status for areas outside established restriction areas. The proposed monitoring schemes potentiate an enormous reduction of sampling costs, but call for a revision of the regulation for the protection against Aujeszky's disease.

p38-avidity-ELISA: examination of herds experiencing epidemic or endemic Neospora caninum-associated bovine abortion.

An enzyme-linked immunosorbent assay (ELISA) was established to measure the avidity of bovine IgG directed against p38, a surface antigen (NCSRS2) of Neospora caninum tachyzoites. In the sera of dams intravenously infected with N. caninum NC-1, the p38-specific avidity increased from initially below 40% up to avidity indices between 50 and 80% after days 23 and 91 p.i. The p38-avidity-ELISA was used to examine various herds that had experienced endemic and epidemic N. caninum-associated bovine abortions. In herds with endemic abortion, generally high avidity indices of N. caninum-specific IgG were detected. This finding and the observation of an association between the seropositivity of dams and that of their offspring suggested a predominantly vertical transmission of the parasite among the animals of these herds, thus indicating chronic infection of these cattle. In contrast, for herds experiencing epidemic abortion: (i) an association regarding seropositivity of dams and their daughters could not be shown and (ii) a generally low avidity of p38-specific IgG in the sera from aborting dams was determined. This indicates recent postnatal transmission of N. caninum in these herds. A linear regression model explaining the avidity of p38-specific IgG was significantly influenced by the time span between sampling and the occurrence of the first abortion of the epidemic in herds with an N. caninum-associated abortion storm. Another factor significantly contributing to the model was the proportion of dams at risk that had aborted in the herds (i.e. the severity of the abortion epidemic). A possible explanation for this observation is that herds experiencing heavy abortion are sampled earlier after the onset of the epidemic than others that have a less severe abortion storm.

[Experiences with follow-up investigations of oral vaccination campaigns against rabies in foxes in Saxony with special emphasis on a standardised serology]. / Erfahrungen mit der Begleitdiagnostik zur oralen Immunisierung der Füchse in Sachsen unter besonderer Berücksichtigung einer standardisierten Tollwutserologie.

An 8-year experience with organisation and standardisation of follow-up investigations within oral vaccination campaigns against rabies in foxes (OVF) in Saxony is summarised. With respect to OVF, the number of diagnostic tests performed during the years 1992-2000 on foxes amounts to a total of 52,226 Fluorescence antibody-(FAT), 7,551 marker-(TC) and 11,645 serological tests. The mean bait-uptake and the mean immunisation rate in foxes ranged between 78-86% and 60-89%, respectively. Based on the seroconversion rates of the years 1997-2000 observed in vaccination areas and in areas where vaccination was already finished, experience with a standardised serology under routine conditions is presented and discussed. Furthermore, recommendations concerning organisation and logistics of sampling are given.

Analysis of sampling strategies to substantiate freedom from disease in large areas.

In this paper, we deal with the strategies of surveys to substantiate freedom from disease for a certain territory. Infection might not be distributed homogeneously. So, a relatively high within-herd prevalence might be observed while the herd-level prevalence is lower. For this situation, we compare various two-stage sample strategies. The calculation of appropriate sample sizes becomes quite complicated. The theoretical generalization of the hypergeometric distribution by Cameron and Baldock [Prev. Vet. Med. 24 (1998) 1] introduces a simple way to evaluate multi-stage sample sizes while regarding real-test properties. We demonstrate the theoretical foundations of these calculations. These principles open up the possibility of optimizing costs or other relevant variables, by choosing the appropriate sample strategy (each of which ensures the same alpha-level for the first stage). In addition, we evaluate the statistical power of the complete strategies under consideration.Furthermore, we apply our theoretical results to a data example of Brucella melitensis. We used the herd-size situation in Germany, characterized by many small sheep holdings and only a few large ones. The consequences of real-test properties on sample sizes and on the applicability of several strategies are discussed.