Two consecutive large outbreaks of Salmonella Muenchen linked to pig farming in Germany, 2013 to 2014: Is something missing in our regulatory framework?

In 2013, raw pork was the suspected vehicle of a large outbreak (n = 203 cases) of Salmonella Muenchen in the German federal state of Saxony. In 2014, we investigated an outbreak (n = 247 cases) caused by the same serovar affecting Saxony and three further federal states in the eastern part of Germany. Evidence from epidemiological, microbiological and trace-back investigations strongly implicated different raw pork products as outbreak vehicles. Trace-back analysis of S. Muenchen-contaminated raw pork sausages narrowed the possible source down to 54 pig farms, and S. Muenchen was detected in three of them, which traded animals with each other. One of these farms had already been the suspected source of the 2013 outbreak. S. Muenchen isolates from stool of patients in 2013 and 2014 as well as from food and environmental surface swabs of the three pig farms shared indistinguishable pulsed-field gel electrophoresis patterns. Our results indicate a common source of both outbreaks in the primary production of pigs. Current European regulations do not make provisions for Salmonella control measures on pig farms that have been involved in human disease outbreaks. In order to prevent future outbreaks, legislators should consider tightening regulations for Salmonella control in causative primary production settings.

Ongoing haemolytic uraemic syndrome (HUS) outbreak caused by sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157, Germany, December 2016 to May 2017.

We report an ongoing, protracted and geographically dispersed outbreak of haemolytic uraemic syndrome (HUS) and gastroenteritis in Germany, involving 30 cases since December 2016. The outbreak was caused by the sorbitol-fermenting immotile variant of Shiga toxin-producing (STEC) Escherichia coli O157. Molecular typing revealed close relatedness between isolates from 14 cases. One HUS patient died. Results of a case-control study suggest packaged minced meat as the most likely food vehicle. Food safety investigations are ongoing.

Outbreak of Cryptosporidium hominis following river flooding in the city of Halle (Saale), Germany, August 2013.

BACKGROUND: During weeks 32-33, 2013, 24 cases of cryptosporidiosis were notified in the city of Halle (annual mean 2008-2012: 9 cases). We investigated the outbreak to identify the source and recommend control measures, considering that between weeks 23-25 the river Saale which flows through the city centre overflowed the floodplain, parts of the city centre and damaged sewage systems. METHODS: We defined a case as a resident of Halle with gastroenteritis, Cryptosporidium-positive stool and disease onset weeks 27 through 47. In a case-control study among kindergarten children, we compared cases and controls regarding environmental exposure, use of swimming pools, zoo visits and tap water consumption 14 days pre-onset or a corresponding 14-days-period (controls) and adjusted for residence. Stool specimens were tested by microscopy and PCR, and Cryptosporidium DNA was sequenced. Samples from public water system, swimming pools and river Saale were examined for Cryptosporidium oocysts (microscopy and PCR). RESULTS: Overall, 167 cases were detected, 40/167 (24%) were classified as secondary cases. First disease onsets occurred during week 29, numbers peaked in week 34 and started to decrease in week 36. Median age was 8 years (range: 0-77). Compared to controls (n = 61), cases (n = 20) were more likely to report visits to previously flooded areas (OR: 4.9; 95%-CI: 1.4-18) and the zoo (OR: 2.6; 95%-CI: 0.9-7.6). In multivariable analysis visits to the floodplain remained the sole risk factor (OR: 5.5; 95%-CI: 1.4-22). Only C.hominis of a single genotype (IbA9G2) was detected in stools. Oocysts were detected in samples from the river, two local lakes and three public swimming pools by microscopy, but not in the public water supply. CONCLUSIONS: Evidence suggests that activities in the dried out floodplain led to infection among children. Secondary transmissions may be involved. Consequently, authorities recommended to avoid playing, swimming and having picnics in the flood-affected area. Health authorities should consider the potential health risks of long-term surviving parasites persisting on flooded grounds and in open waters even several weeks after the flooding and of bathing places close to sewage spill-overs. Preventive measures comprise water sampling (involving parasites), information of the public and prolonged closures of potentially contaminated sites.

Relevance of Campylobacter to public health--the need for a One Health approach.

Campylobacter species belong to the most important foodborne bacteria which cause gastroenteritis in humans in both developed and developing countries. With increasing reporting rates, the public awareness towards Campylobacter infections is growing continuously. This strengthens the necessity to establish intervention measures for prevention and control of thermophilic Campylobacter spp. along the food chain, as in particular poultry and poultry meat represent a major source of human infections. An interdisciplinary One Health approach and a combined effort of all stakeholders are necessary to ultimately reduce the burden of campylobacteriosis cases in humans. Numerous studies point out, however, that at present a complete elimination of Campylobacter in the food chain is not feasible. The present aim should therefore be to establish control measures and intervention strategies to minimize the occurrence of Campylobacter spp. in livestock (e.g. poultry flocks) and to reduce the quantitative Campylobacter burden in animals and foods. To this end, a combination of intervention methods at different stages of the food chain appears most promising. That has to be accompanied by targeted consumer advice and education campaigns to raise the awareness towards Campylobacter infections.

Epidemiology of campylobacteriosis in Germany - insights from 10 years of surveillance.

BACKGROUND: Campylobacteriosis caused by Campylobacter spp. is the most common notifiable bacterial gastrointestinal disease in Germany and a major problem in many other European countries as well. In contrast to other infectious diseases, e.g., salmonellosis, the annual number of notified campylobacteriosis cases has increased in Germany and other European countries from 2001-2010. METHODS: National surveillance data from 2001 through 2010 were the basis of a detailed description of the epidemiological pattern of Campylobacter infections in Germany. Special focus was placed on geographical distribution and time trends of Campylobacter infections as well as the identification of risk groups. RESULTS: In total, 588,308 cases of campylobacteriosis were recorded during the observed time period. The mean annual incidence increased from 67 cases/100,000 population in 2001 to 80/100,000 population in 2010. Almost 92% of the notified Campylobacter infections were acquired in Germany. A seasonal distribution was observed with a large peak in the summer months and a small peak in January. Incidence was highest in children ≤4 years and young adults 20-29 years of age. Especially young children living in rural regions in Germany seemed to be at high risk of Campylobacter infection. CONCLUSIONS: Campylobacter is the leading cause of bacterial gastroenteritis in Germany, and has been of rising public health concern. There is a need for enhanced prevention of Campylobacter infections and the data presented here may contribute to better target prevention measures with focus on identified risk groups such as children and young adults.

Trace-back and trace-forward tools developed ad hoc and used during the STEC O104:H4 outbreak 2011 in Germany and generic concepts for future outbreak situations.

The Shiga toxin-producing Escherichia coli O104:H4 outbreak in Germany in 2011 required the development of appropriate tools in real-time for tracing suspicious foods along the supply chain, namely salad ingredients, sprouts, and seeds. Food commodities consumed at locations identified as most probable site of infection (outbreak clusters) were traced back in order to identify connections between different disease clusters via the supply chain of the foods. A newly developed relational database with integrated consistency and plausibility checks was used to collate these data for further analysis. Connections between suppliers, distributors, and producers were visualized in network graphs and geographic projections. Finally, this trace-back and trace-forward analysis led to the identification of sprouts produced by a horticultural farm in Lower Saxony as vehicle for the pathogen, and a specific lot of fenugreek seeds imported from Egypt as the most likely source of contamination. Network graphs have proven to be a powerful tool for summarizing and communicating complex trade relationships to various stake holders. The present article gives a detailed description of the newly developed tracing tools and recommendations for necessary requirements and improvements for future foodborne outbreak investigations.

Thermal stability of hepatitis E virus assessed by a molecular biological approach.

BACKGROUND: Hepatitis E virus (HEV) is a pathogen of emerging concern in industrialized countries. The consumption of wild boar meat has been identified as one risk factor for autochthonous HEV infections. Only limited information is available about thermal stability of HEV, mainly due to the lack of rapid and efficient cell culture systems for measurement of HEV infectivity. METHODS: A molecular biological method was implemented in order to distinguish disassembled from intact viral particles using RNase treatment followed by quantitative real-time RT-PCR. The method was applied to a wild boar liver suspension containing HEV genotype 3. RESULTS: Time-course analyses indicated that the decline of protected RNA could be described by a biphasic model with an initial decrease followed by a stationary phase. The stationary phase was reached after 1 hour at 4°C, 3 days at 22°C and 7 days at 37°C with log reductions of 0.34, 0.45 and 1.24, respectively. Protected RNA was detectable until the end of the experiments at day 50 or 70. Heat exposure for 1 minute resulted in a log reduction of 0.48 at 70°C and increased with higher temperatures to 3.67 at 95°C. Although HEV infectivity titration by inoculation of the liver suspension onto three cell lines did not succeed, the results of the RNase-based method are in accordance with published cell culture-based data. CONCLUSIONS: Measurement of intact viral particles using the RNase-based method may provide data on the stability of RNA viruses when cell culture-based infectivity titrations are not efficient or not available. The method enables processing of large sample numbers and may be suitable to estimate stability of HEV in different types of food.

Novel hepatitis E virus genotype in Norway rats, Germany.

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E-like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.

Infection of in vivo differentiated human mast cells with hantaviruses.

Increased vascular permeability is a key feature of the pathological symptoms caused by hantaviruses. Here, we analysed the interaction between hantaviruses and mast cells, which regulate vascular homeostasis. In highly purified human skin mast cells increasing amounts of Hantaan (HTNV) and, to a lower extent, Prospect Hill (PHV) virions were produced. Replication was confirmed by the production of viral plus-strand RNA as determined by a virus strand-specific RT-PCR. PHV but not HTNV elicited early expression of beta interferon, MxA, ISG15 and CCL5 consistent to studies with other cell types. The data demonstrate that mature mast cells are permissive to infection with hantaviruses. This interaction might contribute to the development of vascular leakage syndrome.

Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR.

Hepatitis E is a rare human disease in developed countries. It is caused by hepatitis E virus (HEV), which is probably transmitted zoonotically to humans from domestic pigs and wild boars. Multiple reports on the detection of HEV-specific antibodies in rats have suggested the presence of an HEV-related agent; however, infectious virus or a viral genome has not been demonstrated so far. Here, a nested broad-spectrum RT-PCR protocol was developed capable of detecting different HEV types including those derived from wild boar and chicken. Screening of 30 faecal samples from wild Norway rats (Rattus norvegicus) from Hamburg (Germany) resulted in the detection of two sequences with similarities to human, mammalian and avian HEV. Virus particles with a morphology reminiscent of HEV were demonstrated by immunoelectron microscopy in one of these samples and the virus was tentatively designated rat HEV. Genome fragments with sizes of 4019 and 1545 nt were amplified from two samples. Sequence comparison with human and avian strains revealed only 59.9 and 49.9 % sequence identity, respectively. Similarly, the deduced amino acid sequence for the complete capsid protein had 56.2 and 42.9 % identity with human and avian strains, respectively. Inoculation of the samples onto three different permanent rat liver cell lines did not result in detectable virus replication as assayed by RT-PCR with cells of the fifth virus passage. Further investigations are necessary to clarify the zoonotic potential of rat HEV and to assess its suitability to serve in a laboratory rat animal model for human hepatitis E.

Detection of hepatitis E virus in wild boars of rural and urban regions in Germany and whole genome characterization of an endemic strain.

BACKGROUND: Hepatitis E is an increasingly diagnosed human disease in Central Europe. Besides domestic pigs, in which hepatitis E virus (HEV) infection is highly prevalent, wild boars have been identified as a possible source of human infection. In order to assess the distribution of HEV in the wild boar population of Germany, we tested liver samples originating from different geographical regions for the presence of the HEV genome and compared the detected sequences to animal and human HEV strains. RESULTS: A total of 148 wild boar liver samples were tested using real-time RT-PCR resulting in an average HEV detection rate of 14.9% (95% CI 9.6-21.6). HEV was detected in all age classes and all geographical regions. However, the prevalence of HEV infection was significantly higher in rural as compared to urban regions (p < 0.001). Sequencing of the PCR products indicated a high degree of heterogenicity of the detected viruses within genotype 3 and a grouping according to their geographical origin. The whole genome sequence of an HEV isolate (wbGER27) detected in many wild boars in the federal state of Brandenburg was determined. It belongs to genotype 3i and shows 97.9% nucleotide sequence identity to a partial sequence derived from a human hepatitis E patient from Germany. CONCLUSION: The results indicate that wild boars have to be considered as a reservoir for HEV in Germany and that a risk of HEV transmission to humans is present in rural as well as urban regions.

Marked Underreporting of Pertussis Requiring Hospitalization in Infants as Estimated by Capture-Recapture Methodology, Germany, 2013-2015.

BACKGROUND: In Germany, pertussis became notifiable in eastern federal states in 2002 and nationwide in March 2013. Infants are at greatest risk for severe disease, with a high proportion requiring hospitalization. We implemented enhanced hospital-based surveillance to estimate the incidence of pertussis requiring hospitalization among infants in Germany and to determine the proportion of infants hospitalized with pertussis too young to have been vaccinated. METHODS: Enhanced surveillance was implemented within a nationwide hospital surveillance network (ESPED). We defined cases as children less than 1 year of age hospitalized due to laboratory-confirmed pertussis with disease onset from 01/07/2013-30/06/2015. We matched cases to those ascertained in the national statutory notification system, and estimated incidence using capture-recapture methodology. RESULTS: The estimated annual incidence of pertussis requiring hospitalization in infants was 52/100,000 infants (95% confidence interval [CI] 48-57/100,000), with 39% under-reporting to the national notification system. During the two epidemiologic years under-reporting decreased from 46% to 32% and was lower in eastern than western federal states (21% vs. 40%). Within ESPED, 154 of 240 infants (64%) were younger than or still at the age recommended for the first vaccine dose; 55 (23%) could have received one or more vaccine doses. Median length of hospitalization was 9 days (IQR 5-13 days) and 18% required intensive care treatment. CONCLUSIONS: Our study revealed a high burden of pertussis in infants with marked under-reporting, especially in western federal states where notification was only recently established. Strategies for the prevention of severe pertussis.

Incidence of notified Lyme borreliosis in Germany, 2013-2017.

Lyme borreliosis (LB) is the most commonly reported tick-borne disease in Germany. In 9/16 states, notification of erythema migrans (EM), acute neuroborreliosis (NB) and Lyme arthritis (LA) is mandatory. We describe incidence measures, time trends, geographical distribution and frequencies of manifestations to better understand LB epidemiology and target prevention measures. We used cases notified in the 9 states and confirmed by local health offices, 2013-2017, to calculate incidences by time, place and person. Altogether, we observed 56,446 cases. Disease onset peaked yearly in July. Incidence ranged from 26/100,000 (2015) to 41/100,000 (2013) with mean annual incidences 2013-2017 on district level between 0.5/100,000 and 138/100,000. Median age was 54 years with peaks in boys (5-9 years, mean incidence 36/100,000) and women (50-69 years, mean incidence 57/100,000). 95% experienced EM only, 2.7% NB and 2.1% LA. 54% were female, but more men had NB (56%) and LA (53%, p < 0.001). Hospitalisation was recorded for 10% of LA and 71% of NB cases. LB remains an important public health concern in Germany with marked regional variation. To facilitate early diagnosis and treatment, health authorities should raise awareness among physicians and promote prevention strategies among the general population: tick-bite-protection, prompt tick removal and medical consultation.

A combined case-control and molecular source attribution study of human Campylobacter infections in Germany, 2011-2014.

Campylobacter infection is the most commonly notified bacterial enteritis in Germany. We performed a large combined case-control and source attribution study (Nov 2011-Feb 2014) to identify risk factors for sporadic intestinal Campylobacter infections and to determine the relative importance of various animal sources for human infections in Germany. We conducted multivariable logistic regression analysis to identify risk factors. Source attribution analysis was performed using the asymmetric island model based on MLST data of human and animal/food isolates. As animal sources we considered chicken, pig, pet dog or cat, cattle, and poultry other than chicken. Consumption of chicken meat and eating out were the most important risk factors for Campylobacter infections. Additional risk factors were preparation of poultry meat in the household; preparation of uncooked food and raw meat at the same time; contact with poultry animals; and the use of gastric acid inhibitors. The mean probability of human C. jejuni isolates to originate from chickens was highest (74%), whereas pigs were a negligible source for C. jejuni infections. Human C. coli isolates were likely to originate from chickens (56%) or from pigs (32%). Efforts need to be intensified along the food chain to reduce Campylobacter load, especially on chicken meat.

Hepatitis E virus seroprevalence of domestic pigs in Germany determined by a novel in-house and two reference ELISAs.

Autochthonous hepatitis E virus (HEV) infections by zoonotic transmission of genotype 3 (GT3) have been reported increasingly from industrialized countries. In this paper the development and validation of an IgG ELISA for the detection of HEV-specific antibodies in domestic pigs is described. Comparison of the diagnostic value of Escherichia coli-expressed HEV-GT3 capsid protein (CP) derivatives revealed a carboxy-terminal derivative as most suitable. Validation of the in-house assay using a commercially available IgG ELISA revealed a high diagnostic specificity and sensitivity. The average HEV seroprevalence of domestic pigs from Germany and the federal state Baden-Wuerttemberg determined by the in-house test was 42.7% and 50.3%, respectively. The seroprevalence in different districts of Baden-Wuerttemberg ranged from 34.9% to 60%, but from 0% to 100% between different herds. These data were compared to those achieved by two commercially available ELISA kits and an in-house ratHEV-based ELISA. In conclusion, the CP-based in-house test proved sensitive and specific, indicating that the ORF3-encoded protein might be dispensable for diagnostics. The novel assay also allowed a parallel analysis by a homologous ratHEV-derived antigen. Thus, the novel IgG ELISA represents a useful tool for future standardized seroprevalence studies in domestic pigs from Germany and other regions of Europe.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

Prevalence of Hepatitis E virus-specific antibodies in sera of German domestic pigs estimated by using different assays.

Hepatitis E virus is the causative agent of an acute hepatitis in humans. In industrialized countries, autochthonous hepatitis E cases in the past were mainly of undetermined origin, whereupon nowadays some cases may be linked to zoonotic transmission of HEV from pigs and wild boars. In contrast to several European countries the HEV status of German domestic pigs and a possible risk of transmission are unknown so far. Here, a novel peptide-based ELISA was used to detect HEV-specific antibodies in 1072 sera from German domestic pigs resulting in an average seroprevalence of 49.8% indicating widespread HEV infections in these animals. A comparative testing of 321 randomly selected sera revealed a seroprevalence of 64.8% when using a commercially available ELISA and 43.9% for the novel peptide-based ELISA but concordant results were obtained in both tests only for 56.1% of the sera. Additional re-testing of 23 randomly selected sera with a modified commercially available immunoblot revealed discordant results also. The use of different antigens and the measurement of different immunoglobulin classes are considered to be responsible for the observed variations of the results. Though the present study revealed a high seroprevalence of HEV in the German domestic pig population and a potential risk of transmission to humans, the differing results of the tests highlight the necessity of a standardization of serological assays for comparative seroprevalence and longitudinal studies.