Culex torrentium mosquitoes from Germany are negative for Wolbachia.

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high-throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia-induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.

[A Somali refugee with fever and abnormal blood smear]. / Somalischer Flüchtling mit Fieber und auffälligem Blutausstrich.

The increasing migration into Europe may confront clinicians with diseases barely known any more in this part of the world. We present a case of louse-borne relapsing fever in a Somali refugee. Blood smears led to the diagnosis of relapsing fever and DNA sequencing was positive for Borrelia recurrentis. This disease should be considered in all patients with unclear fever and a compatible travel or migration history. Blood smears are employed as the primary diagnostic method. Therapy harbors the danger of a Jarisch-Herxheimer reaction, a complication that may require intensive medical care.

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real-time PCR assays.

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species-specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).

Human case of Onchocerca lupi infection, Germany, August 2014.

Onchocerca lupi, a nematode parasite infecting dogs and cats with a hitherto unknown arthropod vector, is also being recognised as a parasite also responsible for human eye infections. Here we describe a case of human eye infection diagnosed molecularly by nematode 12S rDNA PCR in a German patient who had travelled to Tunisia and Turkey. The patient recovered after treatment with antibiotic and anti-inflammatory therapy.

Mosquitoes (Diptera: Culicidae) of metropolitan Hamburg, Germany.

In Europe, mosquito-related public health concerns are growing due to the increasing spread of invasive mosquito species and the recent emergence of mosquito-borne arboviruses. A vital backbone in the assessment of these issues is detailed knowledge of the mosquito fauna, i.e. regional mosquito inventories. It was therefore decided to intensify nationwide investigations on the occurrence and distribution of mosquitoes in Germany in order to update old records and to detect possible faunal changes. This paper is focussing on a densely populated metropolitan region, the federal state of Hamburg and its adjacent environs, taking two historical baseline inventories into consideration, spanning almost 100 years of mosquito research in Hamburg. In the period between 2010 and 2014, more than 10,000 juvenile, neonate and adult mosquito specimens were sampled and trapped at 105 sites in Hamburg and its environs, of which about 60% have been identified to species level, resulting in a total of 33 recorded species. Of these, Anopheles algeriensis, Culex modestus, Ochlerotatus caspius, Ochlerotatus nigrinus and Ochlerotatus sticticus are new to the area. The most common species in Hamburg are Culex pipiens/torrentium and Ochlerotatus annulipes/cantans. In contrast, two previously common species, Anopheles atroparvus and Ochlerotatus excrucians, were not detected. Despite substantial environmental changes due to reconstruction, urbanisation and renaturation in the Hamburg metropolitan region in recent decades, there has been remarkably little change within the mosquito fauna during the last century.

Dirofilaria repens and Dirofilaria immitis DNA findings in mosquitoes in Germany: temperature data allow autochthonous extrinsic development.

After the repeated demonstration of Dirofilaria repens infections in German dogs, D. repens and Dirofilaria immitis DNA was detected in mosquitoes trapped in 2011, 2012 and 2013 in northeastern and southwestern Germany within the framework of culicid monitoring projects. As temperature is the most important factor dictating the extrinsic development of dirofilarial larvae in the potential vector, data of the German Weather Service (DWD) were analysed for the regions where the positive mosquitoes had been collected. Based on the mean daily temperatures recorded by weather stations most closely to the collection sites of the mosquitoes, it can be concluded that the mosquitoes were trapped in time periods that allowed for the completion of the developmental cycle of the worms in the mosquitoes and a subsequent transmission to a vertebrate host. The results of this study confirm the principal climatic suitability of certain German regions for the establishment of natural dirofilarial transmission cycles. Moreover, the theoretical climatic considerations, together with findings of D. repens infections in German dogs and mosquitoes, strongly suggest that the continuing spread of at least D. repens from its traditional habitats in the Mediterranean has reached southwestern and northeastern Germany.

A case of autochthonous human Dirofilaria infection, Germany, March 2014.

In March 2014, an infection with the nematode Dirofilaria repens was diagnosed in a German citizen in the federal state of Saxony-Anhalt. The patient had developed an itching subcutaneous nodule containing a female worm, which was identified as D. repens by 12S ribosomal ribonucleic acid (rRNA) gene sequencing. Autochthonous human D. repens infections have not been described in Germany so far, but this finding is consistent with the recent detection of D. repens in mosquitoes from east Germany.

[Mosquitoes as vectors for exotic pathogens in Germany]. / Stechmücken als Überträger exotischer Krankheitserreger in Deutschland.

As a result of intensified globalization of international trade and of substantial travel activities, mosquito-borne exotic pathogens are becoming an increasing threat for Europe. In Germany some 50 different mosquito species are known, several of which have vector competence for pathogens. During the last few years a number of zoonotic arboviruses that are pathogenic for humans have been isolated from mosquitoes in Germany including Usutu, Sindbis and Batai viruses. In addition, filarial worms, such as Dirofilaria repens have been repeatedly detected in mosquitoes from the federal state of Brandenburg. Other pathogens, in particular West Nile virus, are expected to emerge sooner or later in Germany as the virus is already circulating in neighboring countries, e.g. France, Austria and the Czech Republic. In upcoming years the risk for arbovirus transmission might increase in Germany due to increased occurrence of new so-called "invasive" mosquito species, such as the Asian bush mosquito Ochlerotatus japonicus or the Asian tiger mosquito Aedes albopictus. These invasive species are characterized by high vector competence for a broad range of pathogens and a preference for human blood meals. For risk assessment, a number of mosquito and pathogen surveillance projects have been initiated in Germany during the last few years; however, mosquito control strategies and plans of action have to be developed and put into place to allow early and efficient action against possible vector-borne epidemics.

Amoebic liver abscess with negative serologic markers for Entamoeba histolytica: mind the gap!

A 38-year-old male German traveller returning from Asia presented with fever, night sweats and abdominal complaints. Abdominal ultrasonography revealed several fast-growing abscesses of the liver. Three blood cultures as well as serologic investigations for the detection of antibodies to Entamoeba histolytica, performed on day 3 and 7 after the onset of clinical symptoms, remained negative. Stool microscopy revealed the presence of amoeba cysts compatible with E. histolytica infection. Taking both the amoebic and bacterial etiology of the abscesses into consideration, the patient was treated with metronidazole and ciprofloxacin followed by paromomycin. Antibodies to E. histolytica tested positive shortly after anti-amoebic therapy was initiated. The patient fully recovered, and ultrasound follow-up showed complete resolution of the abscesses within 50 days. This case leads to the conclusion that amoebic liver abscess should be considered despite negative amoeba serology and that ultrasonography is an important diagnostic tool for the early diagnosis of extraintestinal amoebiasis.

Identification of an epitope on the Entamoeba histolytica 170-kD lectin conferring antibody-mediated protection against invasive amebiasis.

The emergence of multidrug-resistant organisms and the failure to eradicate infection by a number of important pathogens has led to increased efforts to develop vaccines to prevent infectious diseases. However, the nature of the immune response to vaccination with a given antigen can be complex and unpredictable. An example is the galactose- and N-acetylgalactosamine-inhibitable lectin, a surface antigen of Entamoeba histolytica that has been identified as a major candidate in a vaccine to prevent amebiasis. Vaccination with the lectin can induce protective immunity to amebic liver abscess in some animals, but others of the same species exhibit exacerbations of disease after vaccination. To better understand this phenomenon, we used recombinant proteins corresponding to four distinct domains of the molecule, and synthetic peptides to localize both protective and exacerbative epitopes of the heavy chain subunit of the lectin. We show that protective immunity after vaccination can be correlated with the development of an antibody response to a region of 25 amino acid residues of the lectin, and have confirmed the importance of the antibody response to this region by passive immunization studies. In addition, we show that exacerbation of disease can be linked to the development of antibodies that bind to an NH2-terminal domain of the lectin. These findings are clinically relevant, as individuals who are colonized with E. histolytica but are resistant to invasive disease have a high prevalence of antibodies to the protective epitope(s), compared to individuals with a history of invasive amebiasis. These studies should enable us to develop an improved vaccine for amebiasis, and provide a model for the identification of protective and exacerbative epitopes of complex antigens.

Entamoeba ranarum Infection in a Ball Python (Python regius).

The pathogenic Entamoeba species in snakes is widely regarded to be Entamoeba invadens, which can cause severe amoebiasis with up to 100% mortality. In this case report, we describe a ball python (Python regius) that died after short-term weight loss. Necropsy revealed severe necrotizing colitis with large numbers of intralesional Entamoeba trophozoites. Molecular genetic analysis identified these trophozoites as Entamoeba ranarum, a parasite more usually found in amphibians. Furthermore, the extended history revealed that toads (Rhinella marina) had been housed together with the python. This report illustrates the danger of protozoal cross-infections in exotic animals as well as the importance of molecular genetic tools in Entamoeba diagnosis.

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.

OBJECTIVES: Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures. METHODS: Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. RESULTS: D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27). CONCLUSIONS: The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.

A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.

Tropheryma whipplei in children with diarrhoea in rural Ghana.

Tropheryma whipplei has been hypothesized to be able to cause diarrhoea, but data from young children are scarce. In this hospital-based case-control study 534 stool samples of children aged between 2 months and 15 years from rural Ghana were analysed for the presence of T. whipplei. Overall stool prevalence of T. whipplei was high (27.5%). Although there was no difference in T. whipplei carriage overall between cases and controls, cases aged between 0 and 12 months carried T. whipplei in their stool twice as often as controls without diarrhoea. The results from this study may support the hypothesis that T. whipplei can cause diarrhoea in first-time infection.

Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins.

The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.

The interaction of nuclear proteins with essential promoter element of the chicken cardiac myosin light chain 2 gene is involved in muscle-specific transcription.

A quantitative microinjection procedure has been developed to demonstrate muscle-specific transcription of the myosin light chain 2-A (MLC2-A) promoter in differentiated chicken primary breast muscle cells. Nuclear protein binds to the distal region of the required promoter sequence but not to a mutated version of this sequence. The functional significance of this specific DNA-protein interaction for the promoter activity is demonstrated by 'in vivo' competition of microinjected MLC-CAT reporter construct together with excess of synthetic oligonucleotides encompassing the protein binding sites.

Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.

Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.

Induction of the iron-containing superoxide dismutase in Entamoeba histolytica by a superoxide anion-generating system or by iron chelation.

The regulation of superoxide dismutase (SOD) expression was studied in 4 Entamoeba histolytica isolates. In comparison to anaerobic conditions, cultivation of the amoebae in the presence of superoxide radical anions or a ferrous iron chelator revealed substantial increase of SOD expression. Under the different culture conditions, all SOD activity could be exclusively attributed to an iron-containing type (FeSOD). Northern blot analysis revealed that FeSOD expression was regulated on the transcriptional level. Within the 5'-flanking region of the amoebic FeSOD gene, a 19-bp fragment was found with 68% sequence identity to the consensus motif of the binding site for the ferric uptake regulation gene product of Escherichia coli. Electrophoretic mobility shift assays with this 19-bp fragment and with amoebic nuclear extracts revealed specific DNA/protein complex formation. The results indicate that the regulation of E. histolytica FeSOD expression is similar to that of the manganese-containing SOD (MnSOD) of E. coli.

The electrophoretic karyotype of Entamoeba histolytica.

Although knowledge about gene organization and transcription control in the protozoan parasite Entamoeba histolytica has increased substantially during the last few years, questions remain open about ploidy, organization and number of chromosomes in this human pathogen. To get insight into these questions conditions were elaborated to consistently separate E. histolytica chromosomes using pulsed-field gel electrophoresis. Southern blot analyses indicated variations in number and size of homologous chromosomes between various E. histolytica isolates, but only minor differences were observed between clones of a given isolate. Depending on the isolate used 31-35 chromosomes were identified ranging in size from 0.3 to 2.2 megabases. The assignment of 68 independent cDNA probes to the chromosomes of three axenically cultured E. histolytica isolates identified 14 linkage groups, which suggested a haploid genome-size of < or =20 megabases. As single copy probes bound to as many as four chromosome-sized bands, it is most likely that E. histolytica has a functional ploidity of at least 4.