Phaidra - University of Veterinary Medicine Vienna
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Silencing of ultradian rhythms and metabolic depression during spontaneous daily torpor in Djungarian hamsters
No full textUltradian rhythms of metabolism, body temperature and activity are attenuated or disappear completely during torpor in Djungarian hamsters, for all three ultradian periodicities (URsmall, URmedium and URlarge). URsmall and URmedium disappear during entrance into torpor, whereas URlarge disappear later or continue with a low amplitude. This suggests a tight functional link between torpor and the expression of ultradian rhythms, i.e. torpor is achieved by suppression of metabolic rate as well as silencing of ultradian rhythms. Spontaneous torpor is often initiated after an ultradian burst of activity and metabolic rate, beginning with a period of motionless rest and accompanied by a decrease of metabolic rate and body temperature. To extend previous findings on the potential role of the adrenergic system on torpor induction we analysed the influence of the ß3-adrenergic agonist Mirabegron on torpor in Djungarian hamsters, as compared to the influence of the ß-adrenergic antagonist Propranolol. Hamsters were implanted with 10 day release pellets of Mirabegron (0.06 mg day-1) or Propranolol (0.3 mg day-1). Mirabegron transiently supressed and accelerated ultradian rhythms but had no effect on torpor behaviour. Propranolol did not affect torpor behaviour nor the expression of ultradian rhythms with the dosage applied during this study
Isolation and characterization of Leptospira licerasiae in Austrian swine - a first-time case report in Europe
No full textLeptospiraceae comprise a diverse family of spirochetal bacteria, of which many are involved in infectious diseases of animals and humans. Local leptospiral diversity in domestic animals is often poorly understood. Here we describe the incidental detection of Leptospira (L.) licerasiae in an Austrian pig.During an experiment to characterize the pathogenesis of L. interrogans serovar Icterohaemorrhagiae in pigs, cultivation of a urine sample from a non-challenged contact pig resulted in growth of a spirochetal bacterium that tested negative for pathogenic Leptospira (LipL32 gene). PCR, Sanger sequencing and standard serotyping further confirmed that the recovered isolate was clearly different from the challenge strain L. interrogans serovar Icterohaemorrhagiae used in the animal experiment. Whole genome sequencing revealed that the isolate belongs to the species L. licerasiae, a tropical member of the Leptospiraceae, with no prior record of detection in Europe.This is the first report describing the occurrence of L. licerasiae in Europe. Since L. licerasiae is considered to have intermediate pathogenicity, it will be important to follow the geographical distribution of this species and its pathogenic and zoonotic potential in more detail
Screening for oncogenic AF1q expression predicts disease recurrence in gastric cancer patients
No full textAF1q associates with tumor progression and metastases upon WNT signaling. The downstream WNT target CD44 has demonstrated prognostic significance in gastric cancer (GC). This study evaluates the impact of AF1q on tumor stage and survival in GC patients. Immunohistochemical marker expression was analyzed and data were processed to correlation and survival analysis. Out of 182 GC samples, 178 (97.8%) showed moderate to high AF1q expression (p < 0.001), these samples correlated with positive lymph node stage (p = 0.036). In a subgroup analysis of patients with nodal-positive GC (n = 129, 70.9%), enhanced tumoral AF1q expression resulted in impaired recurrence-free survival (RFS, p = 0.030). Enhanced tumoral CD44 expression resulted in impaired disease-specific survival (DSS) in the subgroup of patients with nodal-positive GC (p = 0.031) as well as in the overall GC group (p = 0.005). AF1q demonstrated as an independent prognostic marker for RFS (p = 0.035) and CD44 for DSS (p = 0.036). AF1q has shown potential for prognostication of RFS in GC patients and is predominantly expressed in nodal-positive GC. Testing AF1q provides a possibility of identifying patients with locoregional (and advanced) disease, particularly at risk for disease recurrence. Implementing AF1q into the diagnostic process may facilitate screening, prognosis estimation as well as consideration of preoperative multimodal treatment in patients qualifying for elective upfront surgery
Exploring the occurrence of Listeria in biofilms and deciphering the bacterial community in a frozen vegetable producing environment
No full textThe establishment of Listeria (L.) monocytogenes within food processing environments constitutes a significant public health concern. This versatile bacterium demonstrates an exceptional capacity to endure challenging environmental conditions in the food processing environment, where contamination of food products regularly occurs. The diverse repertoire of stress resistance genes, the potential to colonize biofilms, and the support of a co-existing microbiota have been proposed as root causes for the survival of L. monocytogenes in food processing environments. In this study, 71 sites were sampled after cleaning and disinfection in a European frozen vegetable processing facility, where L. monocytogenes in-house clones persisted for years. L. monocytogenes and L. innocua were detected by a culture-dependent method at 14 sampling sites, primarily on conveyor belts and associated parts. The presence of biofilms, as determined by the quantification of bacterial load and the analysis of extracellular matrix components (carbohydrates, proteins, extracellular DNA) was confirmed at nine sites (12.7%). In two cases, L. innocua was detected in a biofilm. Furthermore, we explored the resident microbial community in the processing environment and on biofilm-positive sites, as well as the co-occurrence of bacterial taxa with Listeria by 16S rRNA gene sequencing. Pseudomonas, Acinetobacter, and Exiguobacterium dominated the microbial community of the processing environment. Using differential abundance analysis, amplicon sequence variants (ASVs) assigned to Enterobacterales (Enterobacter, Serratia, unclassified Enterobacteriaceae) and Carnobacterium were found to be significantly higher abundant in Listeria-positive samples. Several Pseudomonas ASVs were less abundant in Listeria-positive compared to Listeria-negative samples. Acinetobacter, Pseudomonas, Janthinobacterium, Brevundimonas, and Exiguobacterium were key players in the microbial community in biofilms, and Exiguobacterium and Janthinobacterium were more relatively abundant in biofilms. Further, the microbial composition varied between the different areas and the surface materials
Urbanization does not affect red foxes\u27 interest in anthropogenic food, but increases their initial cautiousness
No full textHuman presence and activities have profoundly altered animals\u27 habitats, exposing them to greater risks but also providing new opportunities and resources. The animals\u27 capacity to effectively navigate and strike a balance between risks and benefits is crucial for their survival in the Anthropocene era. Red foxes (Vulpes vulpes), adept urban dwellers, exhibit behavioral plasticity in human-altered environments. We investigated variations in detection frequency on trail cameras and the behavioral responses (explorative, bold, and fearful) of wild red foxes living along an urbanization gradient when exposed to a metal bin initially presented clean and then filled with anthropogenic food. All fox populations displayed an increased interest and similar explorative behavioral responses toward the anthropogenic food source, irrespective of the urbanization gradient. Despite no impact on explorative behaviors, foxes in more urbanized areas initially showed heightened fear toward the empty bin, indicating increased apprehension toward novel objects. However, this fear diminished over time, and in the presence of food, urban foxes displayed slightly reduced fear compared with their less urban counterparts. Our results highlight foxes\u27 potential for adaptability to human landscapes, additionally underscoring the nuanced interplay of fear and explorative behavioral response of populations living along the urbanization gradient
Comparative proteomic profiling of the ovine and human PBMC inflammatory response
No full textUnderstanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research
Behaviour, heart rate variability and surface temperature of calves after hot-iron disbudding or injection of clove oil or isoeugenol under the horn buds
No full textIn recent years, injection of clove oil was investigated as potential alternative to the use of a hot iron for disbudding due to its cytotoxic and anaesthetic properties. Isoeugenol, the isomer of the main component of clove oil, is available as a pure substance and has some advantages as compared to clove oil, but its effects were studied rarely. In this paper we investigated behaviour, heart rate, heart rate variability and surface temperature around the horn bud of 40 calves in four treatments (N = 10/treatment): injection under each horn bud of 1.5 ml clove oil (CLOV), isoeugenol (ISO) or saline (CON), or hot-iron disbudding (BURN) with local anaesthesia and sedation. Behaviour, heart rate (HR) and heart rate variability (HRV) as well as surface temperature around the horn buds were measured before and at different time points after the treatment up to 7 days post treatment; behaviour was also observed during the treatment. Behavioural observations were performed real-time or per video recording. HR and HRV were analysed during undisturbed lying periods and post-treatment changes relative to baseline were calculated. The maximum surface temperature in the region around each of the two horn buds was used for further analysis. LMM, ANOVA or, for behaviour during treatment, non-parametric tests were used for statistical analysis. There was a treatment*time point interaction (LMM, p<0.05) for behaviours associated with pain (ear flicking, head shaking, head scratching) as well as for self-grooming. HR or HRV changes from baseline did not differ between treatments, but sample size was strongly reduced due to lack of data. The development of the maximum surface temperature around the horn buds differed clearly between treatments. Our findings suggest that the injection of clove oil and especially isoeugenol causes less pain compared to the use of a hot iron without analgesia, but more pain compared to the injection of saline solution
Systematic minireview of the craniocervical junction in dogs with and without brachycephaly
No full textTo identify, quantify and compare clinical and concurrent imaging findings of occipital hypoplasia (OH), syringomyelia (SM) and atlanto-occipital overlapping (AO) in dogs with or without brachycephaly.A focused systematic search for literature was performed in the Web of Science™, PubMed and Google Scholar databases. Both authors screened and classified the identified articles using EndNote and appraised the articles using the Critical Appraisal Skills Program checklists. The main clinical and concurrent imaging features were extracted and evaluated for coexistence of OH, SM, AO, and other imaging findings.Thirty-one articles were included in this minireview. For articles focusing on descriptions of OH, SM and AO, 249 dogs had at least one of these conditions, and 3 of these 249 dogs (1%) had coexistence of all three conditions. For articles focusing on descriptions of the dogs, OH, SM, and AO were identified in 552/19/11/11, 574/2/0/6, and 100/0/0/0 small brachycephalic, small non-brachycephalic, large brachycephalic, and large non-brachycephalic breeds, respectively. For all small brachycephalic dogs, the percentages of affected animals were 40% for OH (p = 0.01), 42% for SM (p < 0.01) and 7% for AO (p = 0.033). The number of dogs having AO and clinical symptoms is low (n = 5).OH, SM and AO are more likely to affect small dogs. AO might be limited to small brachycephalic breeds owing to the geometry of the craniocervical junction. Hence, AO alone might not lead to SM. In individual dogs, readers should carefully interpret the clinical relevance of OH or AO in the absence of SM
Flowcytometric data of intermediate-large cell gastrointestinal lymphoma presenting a gross mass in 32 cats - "let them glow in the flow"
No full textGastrointestinal lymphoma is the most common form of lymphoma in domestic cats. Aggressive phenotypes are much less common but do bear and unfavorable prognosis. Immunophenotyping by flow cytometry (FCM) is not systematically performed in these patients, because of difficulties in the acquisition of suitable sample material from the gastrointestinal tract. A multimodal diagnostic approach is recommended to improve identification of subtypes targeting patient tailored therapeutic strategies. The aim of this prospective study was to present results of multicolor FCM immunophenotyping in surgically removed gastrointestinal mass and relate them with histopathology using the World Health Organization (WHO) classification and clonality PCR testing. Thirty-two patients were included. Eight cats (25%) had gastric, 23 (72%) had intestinal lymphoma and 1 (3%) had gastric/jejunal lymphoma. Intestinal lymphoma sites were represented by 18 small intestinal, 4 ileocaecal, 1 large intestinal. All gastric lymphomas were diffuse large B-cell lymphoma (DLBCL). Small intestinal lymphomas were 10 enteropathy associated T-cell lymphoma type I (EATL I), 2 enteropathy associated T-cell lymphoma type II (EATL II), 2 peripheral T-cell lymphoma (PTCL), 3 DLBCL and one DLBCL+EATL II. The most common small intestinal FCM T-cell phenotype was CD3+CD21- CD4-CD8-CD18+ CD5-CD79- in 7/10 EATL I and one EATL II. The most frequent FCM B-cell phenotype was CD3-CD21+ CD4-CD8-CD18+ CD5-CD79+ in 13/17 DLBCL and the DLBCL+EATL II. Clonality PCR results were positive in 87.5% (28/32) of all cases. No cross-lineage rearrangement was observed. IHC and FCM results agreed in 87.5% (28/32) of all cases. When all 3 methods were combined, consistent results were seen in 75% (24/32). This is the first demonstration of a multicolor FCM approach set in context to the gold standard histopathology and clonality testing results
Genomic and transcriptomic analyses of Wohlfahrtia magnifica, a myiasis-causing flesh fly, reveal key targets for potential control programs
No full textPhD Arbeit - Veterinärmedizinische Universität Wien - 2024PhD thesis - University of Veterinary Medicine Vienna - 2024Background
Wohlfahrtia magnifica, a flesh fly, is one of the most important obligatory traumatic myiasis-causing flies, affecting a range of mammals in several European, African, and Asian countries. The infestation of W. magnifica can lead to significant health and welfare problems and substantial economic losses in the field of animal husbandry. However, research on W. magnifica is quite limited, especially at the molecular level.
Aims of the thesis
In light of this limited information, the aims of this thesis revolved around filling the existing knowledge gaps in W. magnifica. First, I aimed to sequence, assemble, and annotate the genome of W. magnifica. This new genome resource provides an essential starting point for many future fundamental and applied research areas. Second, I aimed to investigate the complex dynamics of gene expression throughout the life cycle of W. magnifica to understand how parasitic larvae regulate specific gene expressions to effectively parasitize their hosts. This part of my research will guide the selection of potential targets for vaccines or insecticides aimed at disrupting the establishment of the larvae on or in hosts. Third, I aimed to identify and characterize the critical components for the development of genetically modified strains in W. magnifica. This represents a significant step in the future establishment of genetic control programs designed to address the infestation of W. magnifica.
Results
In our first article, we adopted the strategy of low DNA input library preparation and successfully sequenced the genome of W. magnifica using a single adult specimen as the genetic source. The assembled genome spans 753.99 Mb, with a N50 length of 5.00 Mb, and contains 59.71% repeat elements. The overall RNA-seq alignment rate reached 93.62%, and 98.8% of complete BUSCOs (Benchmarking Universal Single-Copy Orthologs) were identified, indicating the completeness and high quality of the genome. The genome annotation process predicted 16,718 genes and 20,017 mRNA sequences in the genome, with 64.98% of genes functionally annotated using the UniProt/Swiss-Prot database. The phylogenetic analysis demonstrated that W. magnifica has the closest relationship to Sarcophaga bullata, another member of the Sarcophagidae family, followed by Lucilia cuprina, another myiasis-causing fly. The analysis of gene family expansion revealed that the function of the expanded gene families is associated with immunity, insecticide resistance, responses to heat stress, and cuticle development. Furthermore, using the comparative method 45 positively selected genes were identified exhibiting diverse functions. In the second article, we identified a set of 2049 excretory/secretory (ES) proteins in W. magnifica, which play critical roles in parasite-host interactions. Functional annotation indicated that these ES proteins are primarily associated with processes such as cuticle development, peptidase activity, immune responses, and metabolic activities. With the larval samples collected from wounds of the infested host species, Bactrian camel, and the following pupal and adult stages, the analysis of gene expression indicated that the functions of upregulated genes were distributed in cuticle development, proteolysis, and RNA transcription and translation in second stage larvae; peptidase inhibitor activity and nutrient reservoir activity in third stage larvae; cell and tissue morphogenesis and cell and tissue development in pupae; signal perception (a lot of genes implicated in light perception) and behaviors such as feeding, mating, and locomotion in adult flies. The analysis of gene expression related to parasitism revealed that 88 out of 480 peptidase genes, 110 out of 215 cuticle protein genes and 21 heat shock protein (hsp) genes showed a significant upregulation in the parasitic larval stages. Among peptidases, serine peptidases are actively involved in tissue degradation and nutrient acquisition during the parasitic processes of myiasis-causing larvae. Our investigation showed that up to 22.93% (47/205) of serine peptidase genes were highly expressed in the parasitic larval stages, but only 4.39% (9/205) in the pupal stage and 8.78% (18/205) in the adult stage. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin upregulated in the parasitic second-stage larvae and 10 genes in the GO term "nutrient reservoir activity" is dominantly high in the parasitic third-stage larvae. In the third article, we successfully isolated and characterized two pivotal sex-determination genes in W. magnifica, namely Wmtra (W. magnifica transformer) and Wmtra2 (W. magnifica transformer2), whose orthologs have been utilized to develop genetic control approaches in several insect pests. The splicing of Wmtra transcripts exhibited a sex-specific pattern, with the female variant encoding a fully functional protein and the male counterpart yielding a truncated, non-functional polypeptide, which is similar to blow flies such as Lucilia cuprina and Cochliomyia hominivorax. Furthermore, we identified the elements essential for the construction of a Cas9-based homing gene drive in W. magnifica. From sex-biased gene sets, a range of useful resources for the introduction site of the Cas9-gRNA cassette were provided. In addition, an examination of the set of genes exhibiting a bias toward females led to the identification of five potential candidate genes: vasa (vas), nanos (nanos), bicoid (bcd), Bicaudal C (BicC), and innexin5 (inx5). The promoters of these genes can be used to drive Cas9 germline expression. Our further analysis suggested that the promoter from nanos is better suited than others due to its simple structure and successful use for this purpose in other Diptera. We have also identified six U6 RNA genes in W. magnifica; their promoters can serve as potential candidates for regulating gRNA transcription in Cas9-based homing gene drive.
Conclusions
In the first article, we successfully sequenced the genome of W. magnifica from a single fly specimen using the strategy of low DNA input library preparation. This approach holds promise for its applicability to small Diptera flies that are difficult to rear in a laboratory or are amenable to laboratory rearing but present difficulties in inbreeding. The genome of W. magnifica serves as a valuable resource for fundamental research in areas such as molecular biology, biochemistry, and genetics of W. magnifica. Moreover, it opens doors to numerous applications in comparative genomics, transcriptomics, functional genomics, population genetics, and notably, the development of novel control strategies. The second article offers an in-depth investigation into the dynamics of gene expression across the life cycle of W. magnifica, illuminating how the parasitic larvae upregulate specific gene expression to effectively parasitize their hosts. This study not only advances our understanding of the molecular-level parasitic life cycle of W. magnifica but also paves the way for innovative approaches to disrupt larval parasitism in the host. In the third article, we isolated and characterized essential elements for the construction of genetically modified strains in W. magnifica. This represents an important step towards the development of genetic control programs in W. magnifica. In summary, the thesis has shed light on the biology of W. magnifica, contributing to the development of novel control measures against the traumatic myiasis-causing flesh fly, W. magnifica