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The use of serological tests to detect Trichinella infection in domestic and wild animals and in humans has not been standardised yet. This review provides an uniform set of recommendations for the development and use of serological tests to detect circulating antibodies in serum samples. The recommendations are based on the best scientific published information and on the unpublished data from laboratories with a great expertise in this field and represent the official position of the International Commission on Trichinellosis regarding acceptable methods and the evaluation of the sensitivity and specificity. These recommendations are subject to change as new scientific information becomes available.

Abstract Background A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. Results A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria. Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. Conclusions The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.

Abstract Background In August 2006, a case of leptospirosis occurred in an athlete after a triathlon held around Heidelberg and in the Neckar river. In order to study a possible outbreak and to determine risk factors for infection an epidemiological investigation was performed. Methods Participants of the triathlon were contacted by e-mail and were asked to fill out a standardized questionnaire. In addition, they were asked to supply a serum sample for laboratory diagnosis of leptospirosis. A confirmed case patient was defined as a clinical case (i.e. fever and at least one additional symptom suggestive for leptospirosis) with at least two of the following tests positive: ELISA IgM, latex agglutination testing, or microscopic agglutination testing. Rainfall and temperature records were obtained. Results A total of 142 of 507 triathletes were contacted; among these, five confirmed leptospirosis cases were found. Open wounds were identified as the only significant risk factor for illness (p = 0.02). Heavy rains that preceded the swimming event likely increased leptospiral contamination of the Neckar River. Discussion This is the first outbreak of leptospirosis related to a competitive sports event in Germany. Among people with contact to freshwater, the risk of contracting leptospirosis should be considered by health care providers also in temperate countries, particularly in the summer after heavy rains.

Abstract Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Cryptosporidium is a cause of diarrheal infections responsible for a loss of human and animal welfare worldwide. The impact of the parasite is underestimated and the reported sources of infection are diverse, as it occurs in a wide variety of hosts. Wildlife has been reported as a notifiable source, but few studies are available on its occurrence in European wild boar and cervid species. To determine the occurrence of Cryptosporidium in game in Brandenburg, Germany, a molecular survey was conducted during the 2017 to 2020 hunting seasons. A total of 562 fecal samples from wild boar ( Sus scrofa , n  = 262), roe deer ( Capreolus capreolus , n  = 174), red deer ( Cervus elaphus , n  = 62), fallow deer ( Dama , n  = 51) and 13 samples of unspecified species were analyzed for both 18S ribosomal RNA (18S rRNA) and Cryptosporidium oocyst wall protein (COWP) gene sequence regions. PCR results showed that 21.2 % of the samples ( n  = 119/562) were positive for at least one target gene (18S rRNA: n  = 114; COWP: n  = 14), but differences in Cryptosporidium occurrence were observed within species and hunting seasons, with variations ranging from 1.8 % to 41.7 % (roe deer), respectively. Analysis of Sanger sequences of the 18S rRNA and COWP PCR products indicated that the C. sp. deer genotype was predominant in deer (roe deer: 86.7 %, red deer: 66.7 %, fallow deer: 58.8 %), while C. suis and C. scrofarum were mainly detected in wild boar (88.5 %). The human pathogenic species C. parvum was detected in only 1.2 % ( n  = 7) of the samples analyzed, but without a clear indication of a specific wild animal host. The highest Cryptosporidium diversity was found in wild boar and roe deer with five and four different species, respectively. Comparison of the 18S rRNA sequences with the designated reference revealed minor variations at several nucleotide positions in some isolates, possibly indicating evolutionary adaptations and the development of new subtypes. In conclusion, wildlife represents a reservoir for a diverse spectrum of Cryptosporidium species and may thus contribute to their environmental spread and the transmission to humans., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Background: Temperature, precipitation, and humidity are important factors that can influence the spread, reproduction, and survival of pathogens. Climate change affects these factors, resulting in higher air and water temperatures, increased precipitation, or water scarcity. Climate change may thus have an increasing impact on many infectious diseases., Methods: The present review considers those foodborne pathogens and toxins in animal and plant foods that are most relevant in Germany, on the basis of a selective literature review: the bacterial pathogens of the genera Salmonella, Campylobacter and Vibrio , parasites of the genera Cryptosporidium and Giardia , and marine biotoxins., Results: As climate change continues to progress, all infections and intoxications discussed here can be expected to increase in Germany., Conclusions: The expected increase in foodborne infections and intoxications presents a growing public health risk in Germany., Competing Interests: Conflicts of interest The authors declared no conflicts of interest., (© Robert Koch Institute. All rights reserved unless explicitly granted.)

Environmental, hunting and handling factors affect the microbial load of hunted game and the resulting meat products. The aim of this study was to systematically investigate the influence of several factors on the initial microbial load (IML) of game carcasses during the early hunting chain. Eviscerated roe deer body cavities ( n = 24) were investigated in terms of total viable count and the levels of Pseudomonas spp., Lactobacillus spp., Enterobacteriaceae and Escherichia coli ( E. coli ). Furthermore, a risk analysis based on the obtained original IML data, literature search and a Failure Mode and Effects Analysis (FMEA) was performed. The IML could be explained in a regression model by factors including the higher body weight (BW), damaged gastrointestinal tract by the shot, ambient temperature or rain. The levels of Lactobacillus spp. ( p = 0.0472), Enterobacteriaceae ( p = 0.0070) and E. coli ( p = 0.0015) were lower on the belly flap surface when gloves were used during evisceration. The literature search revealed that studies examining influencing factors (IF) on the IML of game carcasses found contradictory effects of the comparable IF on IML. Potential handling failures may lead to a higher IML of game carcasses during the early hunting chain ranked by FMEA. Several handling practices for game carcasses are recommended, such as ensuring efficient cooling of heavier BW carcasses to limit bacterial growth or eviscerating heavier carcasses before lighter ones.

In the past decade, two leptospirosis outbreaks occurred among strawberry harvesters in Germany, with 13, and 45 reported cases respectively. In both outbreaks, common voles (Microtus arvalis) infected with Leptospira kischneri serovar Grippotyphosa were identified as the most likely outbreak source. In an univariate analysis, eating unwashed strawberries was identified as one of the risk factors associated with Leptospira infection. The aim of this study was to evaluate the survival time of L. kirschneri serovar Grippotyphosa on strawberries under varying conditions. Strawberries were spiked with 5x109 of both a laboratory reference strain (strain Moskva V) and an outbreak field strain (94-6/2007) of L. kirschneri serovar Grippotyphosa sequence type 110. Survival times were investigated in a fully crossed design with three incubation times (2h, 4h, 6h and 8h) and three temperatures (15°C, 21°C and 25°C) with three replicated for each condition. A wash protocol was developed and recovered Leptospira were determined by qPCR, dark field microscopy and culturing. Viable L. kirschneri of both the reference strain and the field strain were identified in all samples at 25°C and an incubation time of 2h, but only 1/9 (11%) and 4/9 (44%) of the samples incubated at 15°C were positive, respectively. Both reference and field strain were viable only in 2/9 (22%) at 25° after 6h. After an 8h incubation, viable Leptospira could not be identified on the surface of the strawberries or within the fruit for any of the tested conditions. Based on these results, the exposure risk of consumers to viable Leptospira spp. through the consumption of strawberries bought at the retail level is most likely very low. However, there is a potential risk of Leptospira infection by consumption of strawberries on pick-your-own farms., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is an occupational risk for military personnel and many cases have been reported worldwide. Rodents are the most important maintenance hosts for Leptospira spp. and may infect both animals and humans. To determine the occurrence and identity of pathogenic Leptospira spp. in rodent and shrew populations in German military camps in Afghanistan, we examined 751 animals ( Mus musculus, Cricetulus migratorius, Meriones libycus, Rattus tanezumi, Crocidura cf. suaveolens, and Suncus etruscus) from four military camps in Northern Afghanistan from 2009-12. Leptospiral DNA was found in 1.1% of the animals and only in Mus musculus. Partial secY sequencing identified Leptospira borgpetersenii and Leptospira kirschneri as infecting genomospecies. Multilocus sequence typing was successful in the L. borgpetersenii samples, which were identified as sequence type 155. The low prevalence we observed suggested that the exposure risk of military personnel to infectious Leptospira spp. in the region is low.

Comparison of epidemiological data on the occurrence of Toxoplasma (T.) gondii tissue cysts in meat is hampered by the lack of standardization and a great variety of methods for molecular detection. Therefore, this study aimed to compare and validate three different polymerase chain reaction (PCR) methods for detection of T. gondii DNA in pork. Analytical performance characteristics of two real time PCRs (qPCRs; Tg-qPCR1, Tg-qPCR2) and one conventional endpoint PCR (cPCR), all targeting the 529 repeated element, were assessed using genomic DNA of three clonal T. gondii types prevailing in Europe and North America. qPCR efficiencies for all three clonal types ranged between 93.8 and 94.4% (Tg-qPCR1) and 94.3-95.6% (Tg-qPCR2). Tg-qPCR1 and Tg-qPCR2 showed an overall PCR performance score of 85% and displayed a similar 95% detection limit of 1.067 and 1.561 genome equivalents per PCR reaction (GE/PCR), respectively. However, T. gondii DNA could be detected at concentrations as low as 0.1 GE/PCR. Reliable quantification is possible over 4 log ranges from 10 5 to 100 GE/PCR with mean repeatability relative standard deviations of ≤11% and reproducibility relative standard deviations of ≤12.7%. Presumably, both qPCRs are similarly suitable for sensitive and specific detection of T. gondii DNA in pork. In contrast, the cPCR using primer pair TOX5/Tox-8 proved to be highly sensitive with a detection limit of 1.41 GE/PCR, but not suitable for detection of T. gondii DNA in pork as unspecific amplification of porcine DNA was observed resulting in bands with similar size to the desired T. gondii -specific PCR product., (© 2019 The Authors.)

In this study, the prevalence of Yersinia pseudotuberculosis in wild boars in northeast Germany was determined. For that purpose, the tonsils of 503 wild boars were sampled. The presence of Y. pseudotuberculosis was studied by diagnostic PCR. Positive samples were analyzed by cultural detection using a modified cold enrichment protocol. Ten Y. pseudotuberculosis isolates were obtained, which were characterized by biotyping, molecular serotyping, and multilocus sequence typing (MLST). In addition, whole-genome sequences and the antimicrobial susceptibility of the isolates were analyzed. Yersinia pseudotuberculosis was isolated from male and female animals, most of which were younger than 1 year. A prevalence of 2% (10/503) was determined by cultural detection, while 6.4% (32/503) of the animals were positive by PCR. The isolates belonged to the biotypes 1 and 2 and serotypes O:1a ( n = 7), O:1b ( n = 2), and O:4a ( n = 1). MLST analysis revealed three sequence types, ST9, ST23, and ST42. Except one isolate, all isolates revealed a strong resistance to colistin. The relationship of the isolates was studied by whole-genome sequencing demonstrating that they belonged to four clades, exhibiting five different pulsed-field gel electrophoresis (PFGE) restriction patterns and a diverse composition of virulence genes. Six isolates harbored the virulence plasmid pYV. Besides two isolates, all isolates contained ail and inv genes and a complete or incomplete high-pathogenicity island (HPI). None of them possessed a gene for the superantigen YPM. The study shows that various Y. pseudotuberculosis strains exist in wild boars in northeast Germany, which may pose a risk to humans. IMPORTANCE Yersinia pseudotuberculosis is a foodborne pathogen whose occurrence is poorly understood. One reason for this situation is the difficulty in isolating the species. The methods developed for the isolation of Yersinia enterocolitica are not well suited for Y. pseudotuberculosis We therefore designed a protocol which enabled the isolation of Y. pseudotuberculosis from a relatively high proportion of PCR-positive wild boar tonsils. The study indicates that wild boars in northeast Germany may carry a variety of Y. pseudotuberculosis strains, which differ in terms of their pathogenic potential and other properties. Since wild boars are widely distributed in German forests and even populate cities such as Berlin, they may transmit yersiniae to other animals and crop plants and may thus cause human infections through the consumption of contaminated food. Therefore, the prevalence of Y. pseudotuberculosis should be determined also in other animals and regions to learn more about the natural reservoir of this species., (Copyright © 2018 American Society for Microbiology.)

Ochrobactrum and Brucella are closely related bacteria that populate different habitats and differ in their pathogenic properties. Only little is known about mobile genetic elements in these genera which might be important for survival and virulence. Previous studies on Brucella lysogeny indicated that active phages are rare in this genus. To gain insight into the presence and nature of prophages in Ochrobactrum , temperate phages were isolated from various species and characterized in detail. In silico analyses disclosed numerous prophages in published Ochrobactrum genomes. Induction experiments showed that Ochrobactrum prophages can be induced by various stress factors and that some strains released phage particles even under non-induced conditions. Sixty percent of lysates prepared from 125 strains revealed lytic activity. The host range and DNA similarities of 19 phages belonging to the families Myoviridae, Siphoviridae , or Podoviridae were determined suggesting that they are highly diverse. Some phages showed relationship to the temperate Brucella inopinata phage BiPB01. The genomic sequences of the myovirus POA1180 (41,655 bp) and podovirus POI1126 (60,065 bp) were analyzed. Phage POA1180 is very similar to a prophage recently identified in a Brucella strain isolated from an exotic frog. The POA1180 genome contains genes which may confer resistance to chromate and the ability to take up sulfate. Phage POI1126 is related to podoviruses of Sinorhizobium meliloti (PCB5), Erwinia pyrifoliae (Pep14), and Burkholderia cenocepacia (BcepIL02) and almost identical to an unnamed plasmid of the Ochrobactrum intermedium strain LMG 3301. Further experiments revealed that the POI1126 prophage indeed replicates as an extrachromosomal element. The data demonstrate for the first time that active prophages are common in Ochrobactrum and suggest that atypical brucellae also may be a reservoir for temperate phages.

Virulent phages have been used for many years to type Brucella isolates, but until recently knowledge about the genetic makeup of these phages remains limited. In this work the host specificity and genomic sequences of the original set (deposited in 1960) of VLA Brucella reference phages Tb, Fi, Wb, Bk2, R/C, and Iz were analyzed and compared with hitherto described brucellaphages. VLA phages turned out to be different from homonymous phages in other laboratories. The host range of the phages was defined by performing plaque assays with a wide selection of Brucella strains. Propagation of the phages on different strains did not alter host specificity. Sequencing of the phages Tb V , Fi V , Wb V , and R/C V revealed nucleotide variations when compared to same-named phages previously described by other laboratories. The phages Bk2 V and Iz V were sequenced for the first time. While Bk2 V exhibited the same deletions as Wb V , Iz V possesses the largest genome of all Brucella reference phages. The duplication of a 301 bp sequence in this phage and the large deletion in Bk2 V , Wb V , and R/C V may be a result of recombination caused by repetitive sequences located in this DNA region. To identify new phages as potential candidates for lysotyping, the host range and Single Nucleotide Polymorphisms (SNPs) of 22 non-reference Brucella phages were determined. The phages showed lysis patterns different from those of the reference phages and thus represent novel valuable candidates in the typing set.

Previous research on trichinellosis in Africa focused on isolating Trichinella from wildlife while the role of domestic pigs has remained highly under-researched. Pig keeping in Uganda is historically recent, and evidence on zoonotic pig diseases, including infection with Trichinella species, is scarce. A cross-sectional survey on Trichinella seroprevalence in pigs was conducted in three districts in Central and Eastern Uganda from April 2013 to January 2015. Serum from a random sample of 1125 pigs from 22 villages in Eastern and Central Uganda was examined to detect immunoglobulin G (IgG) against any Trichinella spp. using a commercially available ELISA based on excretory-secretory antigen. ELISA positive samples were confirmed using Western Blot based on somatic antigen of Trichinella spiralis as recommended in previous validation studies. Diaphragm pillar muscle samples (at least 5 g each) of 499 pigs from areas with high ELISA positivity were examined using the artificial digestion method. Overall, 78 of all 1125 animals (6.9%, 95% CI: 5.6-8.6%) tested positive for antibodies against Trichinella spp. in the ELISA at significantly higher levels in Kamuli district compared to Masaka and Mukono districts. Thirty-one percent of the ELISA positive samples were confirmed IgG positive by the Western Blot leading to an overall seroprevalence of 2.1% (95% CI: 1.4-3.2%). The large proportion of ELISA positive samples that could not be confirmed using Western blot may be the result of cross-reactivity with other gastrointestinal helminth infections or unknown host-specific immune response mechanisms in local pig breeds in Uganda. Attempts to isolate muscle larvae for species determination using the artificial digestion method were unsuccessful. Due to the large number of muscle samples examined we are confident that even if pigs are infected, the larval burden in pork is too low to pose a major risk to consumers of developing trichinellosis. This was the first large systematic field investigation of Trichinella infection in domestic pigs in Uganda and its results imply that further studies are needed to identify the Trichinella species involved, and to identify potential sources of infection for humans., Competing Interests: The authors have declared that no competing interests exist.

We analyzed risk factors for Leptospira seropositivity in humans, using data from a population-based cross-sectional zoonosis survey in South Germany (2008/9). Out of 1007 participants 42 (4.2 %) were sero-positive (19/446 men; 23/561 women), indicating that Leptospira exposure and sero-conversion is much more frequent than commonly assumed. Relative risks (RR) for seropositivity with exact 95 % confidence intervals (CI; adjusted for specificity and sensitivity of the ELISA test) were calculated for various exposure factors. Contact with pet rats (RR = 13.9 CI [4.8; 25.3]), guinea pigs (3.0[1.1; 7.4]), cattle (3.7[1.3; 9.6]), poultry (3.6[1.3; 8.6]) or livestock (2.3[1.1; 4.9]) as well as occupation as forestry worker (9.2[2.6; 21.4]) were identified as important exposure factors. None of the participants has ever been diagnosed with leptospirosis, yet 45 had experienced symptoms which may have been caused by Leptospira infection (12 with scleral icterus, 25 dark urine, 8 liver inflammation, 7 kidney failure). Three times as many participants with prior symptoms were seropositive as participants without symptoms (RR = 3.4[1.3; 8.3]), suggesting that sero-positive patients with severe symptoms may frequently not be diagnosed as leptospirosis cases. Physicians should consider leptospirosis as a differential diagnosis. Currently, the vast majority of symptomatic leptospirosis patients may neither be diagnosed nor reported.

A gap in the leptospirosis field remains the lack of well-characterized sample collections that allow for comparison of new methods to standard ones. In the context of a flood-related outbreak of leptospirosis evaluated in Anuradhapura, Sri Lanka, a specimen bank was obtained with detailed metadata accompanied by gold standard diagnostic test results. Blood samples collected on admission and 14 days later from suspected cases of leptospirosis were tested using microscopic agglutination test (MAT) (17 serovars), an in-house enzyme-linked immunosorbent assay (ELISA) using a locally obtained strain of Leptospira kirschneri as sonicated antigen, a commercially available ELISA based on sonicated Leptospira biflexa, and a quantitative polymerase chain reaction (qPCR) assay targeting the pathogenic Leptospira-specific 16S rRNA gene. Of 62 patients presenting within the first 2 days of illness, 31 had confirmed leptospirosis based either on paired-sample MAT or qPCR. During the acute phase, qPCR was most sensitive, detecting 74% of definitively diagnosed cases; immunoglobulin G (IgG) ELISA (in-house), IgG ELISA (commercial), and MAT had sensitivities of 35.5%, 12.0%, and 22.6%, respectively, in detecting definitively diagnosed cases using acute phase serum. Of 40 patients with paired sera, 10 were qPCR positive. Of these, five samples were negative by paired-sample MAT. Of the 11 MAT-positive samples, only five were detected using qPCR confirming that both tests are needed for maximal sensitivity. Regional leptospiral serovar-specific IgG ELISA was superior to MAT. Knowing the regionally dominant serovars improves serological sensitivity in the analysis of acute specimens by ELISA, but qPCR was most sensitive in this patient population., (© The American Society of Tropical Medicine and Hygiene.)

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.

Brucella species are important human and animal pathogens. Though, only little is known about mobile genetic elements of these highly pathogenic bacteria. To date, neither plasmids nor temperate phages have been described in brucellae. We analyzed genomic sequences of various reference and type strains and identified a number of putative prophages residing within the Brucella chromosomes. By induction, phage BiPBO1 was isolated from Brucella inopinata. BiPBO1 is a siphovirus that infects several Brucella species including Brucella abortus and Brucella melitensis. Integration of the phage genome occurs adjacent to a tRNA gene in chromosome 1 (chr 1). The bacterial (attB) and phage (attP) attachment sites comprise an identical sequence of 46 bp. This sequence exists in many Brucella and Ochrobactrum species. The BiPBO1 genome is composed of a 46,877 bp double-stranded DNA. Eighty-seven putative gene products were determined, of which 32 could be functionally assigned. Strongest similarities were found to a temperate phage residing in the chromosome of Ochrobactrum anthropi ATCC 49188 and to prophages identified in several families belonging to the order rhizobiales. The data suggest that horizontal gene transfer may occur between Brucella and Ochrobactrum and underpin the close relationship of these environmental and pathogenic bacteria.

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4(+) T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure to Leptospira). Whole-blood specimens were stimulated with Leptospira antigens in vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4(+) T cells. The production of inflammatory cytokines such as TNF-α by CD4(+) T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

New treatment strategies for inflammatory bowel disease are needed and parasitic nematode infections or application of helminth components improve clinical and experimental gut inflammation. We genetically modified the probiotic bacterium Escherichia coli Nissle 1917 to secrete the powerful nematode immunomodulator cystatin in the gut. This treatment was tested in a murine colitis model and on post-weaning intestinal inflammation in pigs, an outbred model with a gastrointestinal system similar to humans. Application of the transgenic probiotic significantly decreased intestinal inflammation in murine acute colitis, associated with increased frequencies of Foxp3(+) Tregs, suppressed local interleukin (IL)-6 and IL-17A production, decreased macrophage inflammatory protein-1α/β, monocyte chemoattractant protein -1/3, and regulated upon activation, normal T-cell expressed, and secreted expression and fewer inflammatory macrophages in the colon. High dosages of the transgenic probiotic were well tolerated by post-weaning piglets. Despite being recognized by T cells, secreted cystatin did not lead to changes in cytokine expression or macrophage activation in the colon. However, colon transepithelial resistance and barrier function were significantly improved in pigs receiving the transgenic probotic and post-weaning colon inflammation was reduced. Thus, the anti-inflammatory efficiency of a probiotic can be improved by a nematode-derived immunoregulatory transgene. This treatment regimen should be further investigated as a potential therapeutic option for inflammatory bowel disease.

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.

Background: Giardia duodenalis is a common flagellated protozoan parasite that infects the small intestine of a wide range of vertebrate hosts. This study aimed to determine whether tracing of G. duodenalis isolates by current genetic typing tools is possible using an exemplary set of samples from infected cattle, buffalo and children from the Ismailia province, Egypt., Method: A total of 804 fecal samples from ruminant animals was collected from 191 herds and 165 samples from diarrheal children below the age of 10 years. Parasites were detected in these samples using the copro-antigen RIDA®QUICK test and by real-time PCR. Samples were then genetically characterized based on the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes., Results: The prevalence of G. duodenalis was 53% in ruminants and 21% in symptomatic children and infection was not positively correlated with diarrheal symptoms. Sequence typing analysis confirmed predominance of B-type sequences (>67%) in humans and E-type sequences (>81%) in ruminants over A-type sequences. For 39 samples the complete sequence information of the three marker gene fragments could be derived. Integration of the concatenated sequence information of the three marker gene fragments with the spatial data of the respective sample revealed that identical or near identical (only up to 1 out of 1358 bp different) concatenated sequencing types were spatially related in 4 out of 5 cases., Conclusion: The risk of zoonotic infection emanating from ruminants even in high prevalence areas is negligible. Genetic characterization indicated a predominant anthropogenic cycle of infection within the pediatric population studied. Integration of sequence typing data with information on geographic origins of samples allows parasite sub-population tracing using current typing tools.

We report on the 41,143-bp genome of brucellaphage F1, a podovirus that infects several Brucella species. The F1 genome is almost identical to the genome of brucellaphage Tb. However, some structural proteins of the phages exhibit extensive polymorphisms and might be responsible for their different host ranges.

Classical microbiological diagnosis of human brucellosis is time-consuming, hazardous, and subject to variable interpretation. Intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the routine identification of Brucella spp. Analysis of mass peak patterns allowed accurate identification to the genus level. However, statistical models based on peak intensities were needed for definite species differentiation. Interlaboratory comparison confirmed the reproducibility of the results.

Twenty-eight-day-old weaned pigs were fed diets with a low (LZn), medium (MZn), or high (MZn) Zn concentration (50 to 80, 150, or 2,500 mg Zn/kg of diet, respectively) provided as zinc oxide (ZnO)(24 pigs per group). They were infected orally with Salmonella enterica serovar Typhimurium DT104 on day 32. Salmonellae were cultivated from feces (up to 42 days postinfection [dpi]) and organs (2 and 42 dpi). Activation of the adaptive systemic and mucosal immune systems was investigated by recording anti-Salmonella IgG levels and levels of B and T lymphocyte subpopulations in blood and gut-associated lymphatic tissue. Growth performance was recorded as well. Salmonellae were shed at higher levels and for longer periods in the HZn group (P < 0.05), with no differences in the tissues. At 2 dpi, the relative percentages of CD4(+) T helper cells (P < 0.01) and of CD2(+) T and NK cells (P < 0.01) in blood were reduced from the relative cell counts obtained at 0 dpi, irrespective of the Zn group. The lowest percentage of cytotoxic T cells was found 14 dpi in the HZn group relative to the MZn (P < 0.05) and LZn (P < 0.01) groups. Supplementation of the feed with 2,500 mg Zn/kg of diet immediately after weaning could positively affect the immune responses of piglets infected with Salmonella Typhimurium, but for a short period only. After 2 weeks, all positive effects disappeared, and rather negative effects, such as higher shedding of salmonellae, lower T cell frequencies, and worse performance, occurred. Thus, supplementation with ZnO at high levels in the pig industry should be limited to 2 to 3 weeks.

Salmonella enterica serovar Typhimurium DT 104 is the major pathogen for salmonellosis outbreaks in Europe. We tested if the probiotic bacterium Enterococcus faecium NCIMB 10415 can prevent or alleviate salmonellosis. Therefore, piglets of the German Landrace breed that were treated with E. faecium (n = 16) as a feed additive and untreated controls (n = 16) were challenged with S. Typhimurium 10 days after weaning. The presence of salmonellae in feces and selected organs, as well as the immune response, were investigated. Piglets treated with E. faecium gained less weight than control piglets (P = 0.05). The feeding of E. faecium had no effect on the fecal shedding of salmonellae and resulted in a higher abundance of the pathogen in tonsils of all challenged animals. The specific (anti-Salmonella IgG) and nonspecific (haptoglobin) humoral immune responses as well as the cellular immune response (T helper cells, cytotoxic T cells, regulatory T cells, γδ T cells, and B cells) in the lymph nodes, Peyer's patches of different segments of the intestine (jejunal and ileocecal), the ileal papilla, and in the blood were affected in the course of time after infection (P < 0.05) but not by the E. faecium treatment. These results led to the conclusion that E. faecium may not have beneficial effects on the performance of weaned piglets in the case of S. Typhimurium infection. Therefore, we suggest a critical discussion and reconsideration of E. faecium NCIMB 10415 administration as a probiotic for pigs.

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.

Background: A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars., Results: A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria.Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars., Conclusions: The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology laboratories.

In 2008, a Trichinella spp. outbreak occurred on a small family-owned pig farm in Mecklenburg-Western Pomerania in northeastern Germany. To obtain epidemiologic information on this outbreak, we determined that after 2005 the prevalence of Trichinella spp. in wild boars has increased in this region of Germany. We discuss the potential role of the raccoon dog in the increase in Trichinella spp. prevalence in the sylvatic cycle in this region. We believe that this increase could pose a threat to pigs kept in back yard conditions, and we provide recommendations to ensure public health safety.

Background: In August 2006, a case of leptospirosis occurred in an athlete after a triathlon held around Heidelberg and in the Neckar river. In order to study a possible outbreak and to determine risk factors for infection an epidemiological investigation was performed., Methods: Participants of the triathlon were contacted by e-mail and were asked to fill out a standardized questionnaire. In addition, they were asked to supply a serum sample for laboratory diagnosis of leptospirosis. A confirmed case patient was defined as a clinical case (i.e. fever and at least one additional symptom suggestive for leptospirosis) with at least two of the following tests positive: ELISA IgM, latex agglutination testing, or microscopic agglutination testing. Rainfall and temperature records were obtained., Results: A total of 142 of 507 triathletes were contacted; among these, five confirmed leptospirosis cases were found. Open wounds were identified as the only significant risk factor for illness (p = 0.02). Heavy rains that preceded the swimming event likely increased leptospiral contamination of the Neckar River., Discussion: This is the first outbreak of leptospirosis related to a competitive sports event in Germany. Among people with contact to freshwater, the risk of contracting leptospirosis should be considered by health care providers also in temperate countries, particularly in the summer after heavy rains.

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.

The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.

Background: Although leptospirosis is a reemerging zoonosis of global importance, outbreaks related to agricultural exposures are primarily situated in tropical countries. In July 2007, a suspected leptospirosis outbreak was recognized among strawberry harvesters from Eastern Europe who were working in Germany. An investigation was initiated to identify the outbreak source and the risk factors for infection., Methods: We conducted a retrospective cohort study with use of a questionnaire administered to harvesters by health authorities in Romania, Slovakia, and Poland. Collected serum samples were tested by microscopic agglutination test and immunoglobulin M enzyme-linked immunosorbent assay. A case patient was defined as a person who worked in the strawberry field during the period 5 June-8 September 2007 and had leptospirosis-compatible symptoms and either an antibody titer 1:800 and a positive immunoglobulin M enzyme-linked immunosorbent assay result (for a confirmed case) or no serological confirmation (for a suspected case). Local rodents were examined for leptospirosis., Results: Among 153 strawberry harvesters, we detected 13 confirmed case patients who had test results positive for antibodies against Leptospira species serogroup Grippotyphosa and 11 suspected case patients (attack rate, 16%). Risk of disease increased with each day that an individual worked in the rain with hand wounds (odds ratio, 1.1; 95% confidence interval, 1.04-1.14) and accidental rodent contact (odds ratio, 4.8; 95% confidence interval, 1.5-15.9). Leptospires of the serogroup Grippotyphosa were isolated from the kidneys of 7 (64%) of 11 voles., Conclusions: This is, to our knowledge, the largest leptospirosis epidemic to occur in Germany since the 1960s. Contact between hand lesions and contaminated water or soil and infected voles was the most likely outbreak source. The unusually warm winter of 2006-2007 supported vole population growth and contributed to this resurgence of leptospirosis in Germany. Because of ongoing climate change, heightened awareness of leptospirosis in temperate regions is warranted.

Summary: Throughout much of the world, Trichinella spp. are found to be the causative agents of human trichinellosis, a disease that not only is a public health hazard by affecting human patients but also represents an economic problem in porcine animal production and food safety. Due to the predominantly zoonotic importance of infection, the main efforts in many countries have focused on the control of Trichinella or the elimination of Trichinella from the food chain. The most important source of human infection worldwide is the domestic pig, but, e.g., in Europe, meats of horses and wild boars have played a significant role during outbreaks within the past 3 decades. Infection of humans occurs with the ingestion of Trichinella larvae that are encysted in muscle tissue of domestic or wild animal meat. Early clinical diagnosis of trichinellosis is rather difficult because pathognomonic signs or symptoms are lacking. Subsequent chronic forms of the disease are not easy to diagnose, irrespective of parameters including clinical findings, laboratory findings (nonspecific laboratory parameters such as eosinophilia, muscle enzymes, and serology), and epidemiological investigations. New regulations laying down rules for official controls for Trichinella in meat in order to improve food safety for consumers have recently been released in Europe. The evidence that the disease can be monitored and to some extent controlled with a rigorous reporting and testing system in place should be motivation to expand appropriate programs worldwide.

This is the first longitudinal study conducted over the entire 5-month fattening period in pigs to investigate the infection dynamics of Salmonella Typhimurium and the association between antibody response and the prevalence of these bacteria in feces. A total of 16 weaning pigs were infected with Salmonella Typhimurium DT104 followed by clinical examination and blood and fecal sampling until slaughter 138 days postinoculation. To investigate fecal shedding rates and distribution patterns of Salmonella in internal organs regarding premortem stress, one group of swine was transported before slaughter; the other group was slaughtered without being transported. A positive correlation between bacteremia-associated fever and fecal shedding rate was observed, although 69% (11 of 16) of infected pigs had no diarrhea. All animals excreted Salmonella Typhimurium at high levels within 2 weeks postinoculation; thereafter, the number of positive pigs declined and Salmonella shedding became intermittent. In contrast, the proportion of pigs that tested seropositive was higher over the entire fattening period (except during the first 3 weeks postinoculation), revealing the advantage of enzyme-linked immunosorbent assay for Salmonella screening on herd level. Concerning the distribution in internal organs and cross-contamination during slaughter, the highest level of Salmonella was detected in tonsils and jejunal and ileocecal lymph nodes, whereas salmonellae could not be detected in muscle, spleen, and liver. No specific influence of transport-induced stress on Salmonella shedding rates in feces and distribution patterns in organs was observed.

Pepsin powder constitutes a health risk, potentially causing severe allergic reactions to those handling the chemical. A fluid pepsin formulation was produced and tested, first in a preliminary study and then in a ring trial encompassing four European National Reference Laboratories (NRLs). The purpose of each trial was to ascertain and compare the action of pepsin powder with that of the pepsin fluid for digesting meat and liberating encapsulated Trichinella spiralis larvae for subsequent counting. The quality of digestion was furthermore evaluated by assessing the visibility through the digestion fluid and the amount of debris remaining after digestion. For the ring trial, at each laboratory 20 blinded replicate 100-g samples of pork meat containing a known number of encapsulated T. spiralis larvae (0 to 30) were digested by the magnetic stirrer method using either the standard pepsin powder (10 samples) or the pepsin fluid (10 samples). With an average recovery rate of 70 to 80%, all NRLs found the pepsin fluid and pepsin powder to be equally effective. The NRLs also found no difference between the two pepsin formulations with regard to debris remnants or visibility through the digestion fluid. The use of pepsin fluid may therefore constitute an improvement of the digestion procedure for the analysts involved.