Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

Impact of the Revision of European Food Hygiene Legislation and the Introduction of Convenience-based Food on Food Safety in the German Military.

The implementation of the European Food Regulation in the German military started in 2003 and was fully implemented in 2006. In addition, in 2003 the German military introduced the concept of using convenience-based foods targeted to improve the safety of food served to the troops. The aim of this study was to evaluate the impact of these changes on food safety and the occurrence of food-borne disease outbreaks in the German military. For this purpose, data from a total of 517 food-borne outbreaks that occurred between 1995 and 2019 in the responsible areas of the German military both within the country and abroad were subjected to a retrospective analysis. As a result, a significant decrease (p = 2.47 × 10-5) in the number of the food-borne outbreak was observed in the second observation period (2003-2019) compared to the first period (1995-2002). Food groups often found contaminated with pathogens were desserts and prepared dishes (first period), fresh produce, soups, and sauces (second period). Bacillus cereus, Enterobacteriaceae, Salmonella spp., and Staphylococcus aureus were dominant pathogens isolated from suspected foods during disease outbreaks in both periods, however, the absolute number of isolates reduced significantly in the second period. Therefore it can be concluded that the implementation of European food hygiene regulations together with the introduction of convenience-based foods had a significant positive impact on food safety in the German military.

Prolonged SARS-CoV-2 Infection and Intra-Patient Viral Evolution in an Immunodeficient Child.

BACKGROUND: With the progression of the Coronavirus disease pandemic, the number of mutations in the viral genome has increased, showing the adaptive evolution of severe acute respiratory syndrome coronavirus 2 in humans and intensification in transmissibility. Long-term infections also allow the development of viral diversity. In this study, we report the case of a child with severe combined immu presenting a prolonged severe acute respiratory syndrome coronavirus 2 infection. We aimed to analyze 3 naso-oropharyngeal swab samples collected between August and December 2021 to describe the amino acid changes present in the sequence reads that may have a role in the emergence of new viral variants. METHODS: The whole genome from clinical samples was sequenced through high throughput sequencing and analyzed using a workflow to map reads and then find variations/single-nucleotide polymorphisms. In addition, the samples were isolated in cell culture, and a plaque forming units assay was performed, which indicates the presence of viable viral particles. RESULTS: The results obtained showed that the virus present in all samples is infectious. Also, there were 20 common mutations among the 3 sequence reads, found in the ORF1ab and ORF10 proteins. As well, a considerable number of uncommon mutations were found. CONCLUSIONS: In conclusion, we emphasize that genomic surveillance can be a useful tool to assess possible evolution signals in long-term patients.

Anaplasma phagocytophilum and Babesia Species of Sympatric Roe Deer (Capreolus capreolus), Fallow Deer (Dama dama), Sika Deer (Cervus nippon) and Red Deer (Cervus elaphus) in Germany.

(1) Background: Wild cervids play an important role in transmission cycles of tick-borne pathogens; however, investigations of tick-borne pathogens in sika deer in Germany are lacking. (2) Methods: Spleen tissue of 74 sympatric wild cervids (30 roe deer, 7 fallow deer, 22 sika deer, 15 red deer) and of 27 red deer from a farm from southeastern Germany were analyzed by molecular methods for the presence of Anaplasma phagocytophilum and Babesia species. (3) Results: Anaplasma phagocytophilum and Babesia DNA was demonstrated in 90.5% and 47.3% of the 74 combined wild cervids and 14.8% and 18.5% of the farmed deer, respectively. Twelve 16S rRNA variants of A. phagocytophilum were delineated. While the infection rate for A. phagocytophilum among the four cervid species was similar (71.4% to 100%), it varied significantly for Babesia between roe deer (73.3%), fallow deer (14.3%), sika deer (27.3%) and red deer (40.0%). Deer ≤2 years of age tested significantly more often positive than the older deer for both A. phagocytophilum and Babesia species. (4) Conclusions: This study confirms the widespread occurrence of A. phagocytophilum and Babesia species in wild cervids and farmed red deer in Germany and documents the co-occurrence of the two tick-borne pathogens in free-ranging sika deer.

Draft Anaplasma phagocytophilum Genome Sequences from Five Cows, Two Horses, and One Roe Deer Collected in Europe.

Anaplasma phagocytophilum is a zoonotic tick-borne intracellular bacterium responsible for granulocytic anaplasmosis. As it is difficult to isolate and cultivate, only 20 A. phagocytophilum genomes have been sequenced to date. Here, we present eight A. phagocytophilum genome sequences obtained using alternative approaches based on sequence capture technology.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Amnion-derived mesenchymal stromal cells show a mesenchymal-epithelial phenotype in culture.

The amnionic membrane is a rich source of multipotent mesenchymal stromal cells (hAMSC), which are readily available and show a potential use in regenerative medicine and tissue engineering. Before these cells can be applied clinically, careful characterization is necessary, especially as primary cells are known to change their phenotype in culture. We analyzed the mesenchymal phenotype of hAMSC at different stages after isolation using immunohistochemistry. Shortly after isolation (1 day), 92 % (± 7 %) of the hAMSC expressed the mesenchymal marker vimentin, 2 % (± 1 %) stained for the epithelial marker cytokeratin-7 and 5 % (± 4 %) co-expressed these markers. After 5 days, the double positive cells slightly increased to 7 % (± 3 %), while exclusive expression of cytokeratin-7 or vimentin remained unchanged (1 % ± 2 % and 92 % ± 1 %, respectively). After the first passage, all attached cells were vimentin-positive, while 54 % (± 9 %) co-expressed cytokeratin-7 and vimentin. Thus, we conclude that under culture, hAMSC adopt a hybrid mesenchymal-epithelial phenotype. It is also essential to perform microscopical examination during the first days after isolation to detect contaminations with human amnion-derived epithelial cells in cultures of hAMSC.

Oxygen modulates the response of first-trimester trophoblasts to hyperglycemia.

Pregestational diabetes retards early embryonic growth. Placental and fetal growth are closely associated, suggesting that placental growth is also impaired. During the first trimester of gestation, oxygen tension rises steeply, leading to excessive production of reactive oxygen species (ROS), which is exacerbated in diabetes and may affect placental development. We hypothesized that oxygen modifies hyperglycemic effects on ROS formation, resulting in decreased first-trimester trophoblast growth. This was tested using a first trimester trophoblast-derived cell line (ACH-3P). Normoglycemia did not alter ACH-3P proliferation at 2.5%, 8%, and 21% oxygen. Hyperglycemic conditions for up to 3 days reduced cell number by 65% and resulted in cell cycle (G(1)- and S-phase) changes but only at 21% oxygen. Proliferation reduction could be partially restored by an inhibitor of mitogen-activated protein kinase (MAPK) ERK1/2 but not of Akt/PkB. Intracellular ROS elevation under hyperglycemia was oxygen independent, whereas mitochondrial superoxide levels were enhanced under hyperglycemia only at 21% oxygen. Intervention to modulate cytosolic and mitochondrial ROS, using ROS formation inducers and inhibitors, did not alter cell growth under hyperglycemia at 21% oxygen. The combination of hyperglycemia and high oxygen levels (21%) reduces proliferation of human first-trimester trophoblasts in a ROS-independent manner involving MAPK. This may account for reduced placental growth and, therefore, also for embryonic growth during the first-trimester pregestational diabetic pregnancies when the oxygen tension increases.

Amnion-derived mesenchymal stromal cells show angiogenic properties but resist differentiation into mature endothelial cells.

Mesenchymal stromal cells derived from the human amnion (hAMSC) currently play an important role in stem cell research, as they are multipotent cells that can be isolated using noninvasive methods and are immunologically tolerated in vivo. The objective of this study was to evaluate their endothelial differentiation potential with regard to a possible therapeutic use in vascular diseases. hAMSC were isolated from human term placentas and cultured in Dulbecco's modified Eagle's medium (DMEM) (non-induced hAMSC) or endothelial growth medium (EGM-2) (induced hAMSC). Induced hAMSC changed their fibroblast-like toward an endothelial-like morphology, and were able to take up acetylated low-density lipoprotein and form endothelial-like networks in the Matrigel assay. However, they did not express the mature endothelial cell markers von Willebrand factor and vascular endothelial-cadherin. Gene expression analysis revealed that induced hAMSC significantly downregulated pro-angiogenic genes such as tenascin C, Tie-2, vascular endothelial growth factor A (VEGF-A), CD146, and fibroblast growth factor 2 (FGF-2), whereas they significantly upregulated anti-angiogenic genes such as serpinF1, sprouty1, and angioarrestin. Analysis of protein expression confirmed the downregulation of FGF-2 and Tie-2 (27%±8% and 13%±1% of non-induced cells, respectively) and upregulation of the anti-angiogenic protein endostatin (226%±4%). Conditioned media collected from hAMSC enhanced viability of endothelial cells and had a stabilizing effect on endothelial network formation as shown by lactate dehydrogenase and Matrigel assay, respectively. In summary, endothelial induced hAMSC acquired some angiogenic properties but resisted undergoing a complete differentiation into mature endothelial cells by upregulation of anti-angiogenic factors. Nevertheless, they had a survival-enhancing effect on endothelial cells that might be useful in a variety of cell therapy or tissue-engineering approaches.

Primary bovine colonic cells: a model to study strain-specific responses to Escherichia coli.

The parasitic or commensal lifestyle of bacteria in different hosts depends on specific molecular interactions with the respective host species. In vitro models to study intestinal bacteria-host interactions in cattle are not available. Bovine primary colonocyte (PC) cultures were generated from colon crypt explants. Up to day 4 of culture, the vast majority of cells were of epithelial phenotype (i.e., expressed cytokeratin but not vimentin). PCs harboured mRNA specific for Toll-like receptors (TLR) 1, TLR3, TLR4 and TLR6 but not for TLR2, TLR5, TLR7, TLR8, TLR9 and TLR10. Six hours after inoculation of PC cultures with Escherichia coli (E. coli) prototype strains representing different pathovars (enterohaemorrhagic E. coli [EHEC], enteropathogenic E. coli [EPEC], enterotoxic E. coli [ETEC]), bacteria were found attached to the cells. EPEC adhesion was accompanied by intracellular actin accumulation. An attenuated laboratory strain (E. coli K12 C600) and a bovine commensal E. coli strain (P391) both did not adhere. Bacterial or LPS challenge of PC cultures resulted in specific increases in mRNA transcripts for IL-8, GRO-alpha, MCP-1, RANTES, and IL-10. The level of mRNA transcripts for TGF-beta stayed constant, while IL-12 mRNA was not detectable. Short-term cultures of PCs, maintaining epithelial cell properties, interacted with commensal and pathogenic bacteria in a strain-specific manner and have proven to be a useful in vitro model to study the interaction of bacteria with the bovine intestinal mucosa.

Maternally and naturally acquired antibodies to Shiga toxins in a cohort of calves shedding Shiga-toxigenic Escherichia coli.

Calves become infected with Shiga toxin-producing Escherichia coli (STEC) early in life, which frequently results in long-term shedding of the zoonotic pathogen. Little is known about the animals' immunological status at the time of infection. We assessed the quantity and dynamics of maternal and acquired antibodies to Shiga toxins (Stx1 and Stx2), the principal STEC virulence factors, in a cohort of 27 calves. Fecal and serum samples were taken repeatedly from birth until the 24th week of age. Sera, milk, and colostrums of dams were also assessed. STEC shedding was confirmed by detection of stx in fecal cultures. Stx1- and Stx2-specific antibodies were quantified by Vero cell neutralization assay and further analyzed by immunoblotting. By the eighth week of age, 13 and 15 calves had at least one stx(1)-type and at least one stx(2)-type positive culture, respectively. Eleven calves had first positive cultures only past that age. Sera and colostrums of all dams and postcolostral sera of all newborn calves contained Stx1-specific antibodies. Calf serum titers decreased rapidly within the first 6 weeks of age. Only five calves showed Stx1-specific seroconversion. Maternal and acquired Stx1-specific antibodies were mainly directed against the StxA1 subunit. Sparse Stx2-specific titers were detectable in sera and colostrums of three dams and in postcolostral sera of their calves. None of the calves developed Stx2-specific seroconversion. The results indicate that under natural conditions of exposure, first STEC infections frequently coincide with an absence of maternal and acquired Stx-specific antibodies in the animals' sera.