Anne Mayer-Scholl

Advancement of a multiplex PCR for the differentiation of all currently described Brucella species

To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species. This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis.

Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera

Human trichinellosis is a foodborne disease caused by ingestion of infective Trichinella muscle larvae via pork or meat of other food animals which are susceptible to this zoonotic parasite. There are new approaches for a risk-oriented meat inspection for Trichinella in pigs which are accompanied by monitoring programmes on herd level to control freedom from this parasite. For this purpose, testing schemes utilizing serological tests with a high sensitivity and specificity are required. This study aimed at the evaluation of an ELISA and a Western Blot (WB) for the detection of anti-Trichinella-IgG in terms of sensitivity and specificity taking results of artificial digestion as gold standard. For this purpose, 144 field sera from pigs confirmed as Trichinella-free as well as 159 sera from pigs experimentally infected with T. spiralis (123), T. britovi (19) or T. pseudospiralis (17) were examined by ELISA (excretory-secretory antigen) and WB (crude worm extract). Sera from pigs experimentally infected with four other nematode species were included to investigate the cross-reactivity of the antigen used in the WB. For all Trichinella-positive pig sera, band pattern profiles were identified in the WB and results were analysed in relation to ELISA OD% values. Testing of pig sera revealed a sensitivity of 96.8% for the ELISA and 98.1% for the WB whereas the methods showed a specificity of 97.9 and 100%, respectively. WB analysis of Trichinella-positive pig sera revealed five specific band patterns of 43, 47, 61, 66, and 102 kDa of which the 43 kDa protein was identified as the predominant antigen. The frequency of the band pattern profile was irrespective of the dose and the period of infection as well as the Trichinella species investigated. In conclusion, monitoring in swine farms for Trichinella antibodies should be based on screening pig sera by means of ELISA followed by confirmatory testing through WB analysis.

Survival of Brucella spp. in mineral water, milk and yogurt

Knowledge of the number of organisms in a food product at the time of consumption is crucial to assess the risk from a deliberate contamination of food samples with Brucella. To date, very little data on the survival times of Brucella in different food matrices is available. This study was conducted to assess the survival times of Brucella spp. in water, milk and yogurt. These food products were inoculated with bacteria, serial dilutions of the food samples plated and the number of surviving bacteria counted. Under normal storage conditions Brucella survived in UHT milk for 87 days, for 60 days in water and less than a week in yogurt. Also, when milk was inoculated with low bacterial numbers, Brucella multiplied by five log units within three weeks. Further we could not confirm that a high fat content in food has a protective effect on Brucella survival. Brucella survived in 3.5% and 10.0% fat yogurt for four and two days, respectively. These results show that appropriate methods for the rapid detection of this pathogen from food matrices are required.

Leptospira spp. in Rodents and Shrews in Germany

Leptospirosis is an acute, febrile disease occurring in humans and animals worldwide. Leptospira spp. are usually transmitted through direct or indirect contact with the urine of infected reservoir animals. Among wildlife species, rodents act as the most important reservoir for both human and animal infection. To gain a better understanding of the occurrence and distribution of pathogenic leptospires in rodent and shrew populations in Germany, kidney specimens of 2973 animals from 11 of the 16 federal states were examined by PCR. Rodent species captured included five murine species (family Muridae), six vole species (family Cricetidae) and six shrew species (family Soricidae). The most abundantly trapped animals were representatives of the rodent species Apodemus flavicollis, Clethrionomys glareolus and Microtus agrestis. Leptospiral DNA was amplified in 10% of all animals originating from eight of the 11 federal states. The highest carrier rate was found in Microtus spp. (13%), followed by Apodemus spp. (11%) and Clethrionomys spp. (6%). The most common Leptospira genomospecies determined by duplex PCR was L. kirschneri, followed by L. interrogans and L. borgpetersenii; all identified by single locus sequence typing (SLST). Representatives of the shrew species were also carriers of Leptospira spp. In 20% of Crocidura spp. and 6% of the Sorex spp. leptospiral DNA was detected. Here, only the pathogenic genomospecies L. kirschneri was identified.

Increased prevalence of Trichinella spp., Northeastern Germany, 2008.

Migration of raccoon dogs from Poland may have caused this change.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the hosts immune system, the impact of the host-pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

Distribution of Leptospira Serogroups in Dogs from Berlin, Germany

Leptospirosis is a bacterial zoonosis in which dogs can act as a reservoir for human infection. The annual vaccination of dogs can prevent leptospirosis caused by serovars included in the vaccine. To date, all available vaccines in Germany include only the serovars Icterohaemorrhagiae and Canicola, the most commonly found serovars prior to the introduction of the leptospirosis vaccines. Yet, the involvement of additional serovars in the clinical presentation of leptospirosis in dogs has been described. The objective of this sero-epidemiological study was to examine the different Leptospira serovars currently circulating in a population of dogs suspicious for leptospirosis from Berlin. In 329 dogs presenting at the Small Animal Clinic in Berlin, the predominant serogroup was Australis (24%), followed by Grippotyphosa (20%) and Pomona (9%). A total of 18% of the dogs were diagnosed with clinical leptospirosis; here the most prevalent serogroups were also Australis (28%), Grippotyphosa (18%), and Pomona (14%). The serovar prevalence data presented here confirm that a change of pattern of infecting Leptospira serovars in dogs has taken place in Berlin. This data corresponds to further sero-epidemiological studies from other regions in Germany. To ensure human and canine health, available vaccines should be adapted to include the most important circulating serovars.

Characterization of illegal food items and identification of foodborne pathogens brought into the European Union via two major German airports

Foods of animal origin brought illegally from third party countries into the European Community pose a risk for the introduction of diseases. This can lead to animal disease outbreaks with significant economic and social costs and subsequent severe trade restrictions. Further, disease outbreaks in humans due to illegally imported foods of animal origin have been described, yet, there are very few studies examining the potential human health impact. Passenger baggage is the most likely route by which illegal products enter a country. Therefore, the volume and geographic origin of foods of animal origin introduced illegally into Germany via the Frankfurt International Airport and Berlin-Schönefeld Airport by passenger luggage were characterized. Further, the occurrence of foodborne zoonotic bacteria such as Salmonella spp., Listeria spp., Campylobacter spp., Yersinia spp., Verocytotoxin-producing Escherichia coli (VTEC) and Brucella spp. and the microbial quality of the foods were analysed by total bacterial count. Between 2012 and 2013, a total of 663 food items were seized from 296 passengers arriving in Germany from 35 different departure countries. The majority of confiscates (51%) originated from Turkey and Russia. A selection of 474 samples was subjected to microbiological analyses. Twenty-three food products tested positive for at least one of the pathogens analysed. The majority of the contaminated foods were meat (33%) or meat products (42%), and milk products (21%). Considering that only a small fraction of arriving passengers is subjected to airport custom controls and only a small number of confiscated foods could be analysed during this study, further investigations are needed to understand the public health risks posed by illegally introduced food items.

Trichinella nativa in red foxes (Vulpes vulpes) of Germany and Poland: Possible different origins

In Germany and Poland, the high population density of the red fox (Vulpes vulpes) is considered a public health risk since this wild canid is one of the main reservoirs of Trichinella spp. In 2010 in Poland, a program to monitor the prevalence of Trichinella spp. in the red fox population was launched. After two years, Trichinella spp. larvae were detected in 44 (2.7%) out of 1634 foxes tested. In Germany in the period 2002-2011, Trichinella spp. larvae were in 27 foxes. The Trichinella species detected were: T. spiralis in 15 foxes from Germany (one co-infection with Trichinella britovi and one with Trichinella pseudospiralis) and in 9 foxes from Poland; T. britovi in 8 and 32 foxes from Germany and Poland, respectively; and T. pseudospiralis in 1 fox from Germany. The arctic species Trichinella nativa was detected in 3 foxes from Germany (one co-infection with Trichinella spiralis) and in 1 fox from Poland. The detection of T. nativa outside its known distribution area opens new questions on the ability of this Trichinella species to colonize temperate regions.

Molecular Epidemiology of Brucella Genotypes in Patients at a Major Hospital in Central Peru

ABSTRACT The multiple-locus variable-number repeat analysis of 90 human Brucella melitensis isolates from a large urban area in central Peru revealed variations at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) out of 16 loci investigated, of which 1 (Bruce42) also is used for species identification. Ten genotypes were identified, separated by the number of Bruce42 repeats into two groups that may have distinct phenotypic characteristics. Whereas genotypes with five or six Bruce42 repeats were cultured mainly from adult patients, genotypes with three Bruce42 repeats were isolated from children and young adolescents as well as from adults. In addition, the isolates with three Bruce42 repeats were obtained more often from patients with splenomegaly (P = 0.02) or hepatomegaly (P = 0.006). An annual variation in the diversity of genotypes was observed, possibly reflecting changes in sources of fresh dairy products, supply routes to city shops and markets, and the movement of infected dairy goat herds.