Blood sampling is a challenging procedure in many captive animals. Although manual restraint or anesthesia are usually possible, they entail intense stress and a high risk of injuries or organ failure. Blood sampling using medicinal leeches (Hirudo medicinalis) represents a promising non-invasive alternative to venipuncture; however, leech blood meal was to date used only for qualitative analyses such as genetic or serological screenings. Hence, the aim of this study was to evaluate the suitability of the leech blood sampling method for quantification of hematological and biochemical parameters. Medicinal leeches were manually applied on 67 zoo animals of eleven species, and control blood samples were obtained by venipuncture of the jugular vein. The leeches drew up to 20 ml of blood in 20 to 55 min. Although most hematological and biochemical parameters were significantly altered in leech-derived samples, their values showed strong (r = 0.62-0.79; 10/24 parameters) to very strong (r > 0.8; 13/24 parameters) correlations with venipuncture in all blood parameters, except for sodium (r = 0.39). As the parameter alterations and correlations were similar among species, simple cross-species regression formulas were sufficient to correct the alterations, thereby ensuring good repeatability between leeches and venipuncture in most parameters. Our data thus suggest that medicinal leeches can be used as a reliable non-invasive and stress-reducing alternative to standard venipuncture, even for quantitative assays. This opens new opportunities for a significant improvement to animal welfare in zoological gardens, conservation programmes, and ecophysiological research, where quantification of blood parameters is often needed.
- Keywords
- Hirudo medicinalis, biochemistry, hematology, medicinal leech, non-invasive blood sampling,
- Publication type
- Journal Article MeSH
In accordance with the 3 Rs principle (to replace, reduce and refine) animal models in biomedical research, we have developed and applied a new approach for sampling and analyzing hair follicles in various experimental settings. This involves use of a convenient device for non-invasive collection of hair follicles and processing methods that provide sufficient amounts of biological material to replace stressful and painful biopsies. Moreover, the main components of hair follicles are live cells of epithelial origin, which are highly relevant for most types of malignant tumors, so they provide opportunities for studying aging-related pathologies including cancer. Here, we report the successful use of the method to obtain mouse hair follicular cells for genotyping, quantitative PCR, and quantitative immunofluorescence. We present proof of concept data demonstrating its utility for routine genotyping and monitoring changes in quality and expression levels of selected proteins in mice after gamma irradiation and during natural or experimentally induced aging. We also performed pilot translation of animal experiments to human hair follicles irradiated ex vivo. Our results highlight the value of hair follicles as biological material for convenient in vivo sampling and processing in both translational research and routine applications, with a broad range of ethical and logistic advantages over currently used biopsy-based approaches.
- Keywords
- DNA damage response, cellular senescence, hair follicle cells, irradiation, non-invasive sampling,
- MeSH
- Fluorescent Antibody Technique MeSH
- Genotyping Techniques MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Tail pathology MeSH
- DNA Damage * radiation effects MeSH
- Aging pathology physiology MeSH
- Hair Follicle anatomy & histology metabolism physiology radiation effects MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable disruption worldwide. For efficient SARS-CoV-2 detection, new methods of rapid, non-invasive sampling are needed. This study aimed to investigate the stability of SARS-CoV-2 in a novel medium for gargle-lavage (GL) self-sampling and to compare the performance of SARS-CoV-2 detection in paired self-collected GL and clinician-obtained nasopharyngeal swab (NPS) samples. The stability study for SARS-CoV-2 preservation in a novel medium was performed over 14 days (4 °C, 24-27 °C, and 37 °C). In total, 494 paired GL and NPS samples were obtained at the University Hospital in Olomouc in April 2021. SARS-CoV-2 detection in paired samples was performed with a SARS-CoV-2 Nucleic Acid Detection Kit (Zybio, Chongqing Municipality, Chongqing, China), an Elecsys® SARS-CoV-2 Antigen assay (Roche Diagnostics, Mannheim, Germany), and a SARS-CoV-2 Antigen ELISA (EUROIMMUN, Lübeck, Germany). The stability study demonstrated excellent SARS-CoV-2 preservation in the novel medium for 14 days. SARS-CoV-2 was detected in 55.7% of NPS samples and 55.7% of GL samples using rRT-PCR, with an overall agreement of 91.9%. The positive percent agreement (PPA) of the rRT-PCR in the GL samples was 92.7%, and the negative percent agreement (NPA) was 90.9%, compared with the NPS samples. The PPA of the rRT-PCR in the NPS and GL samples was 93.2% when all positive tests were used as the reference standard. Both antigen detection assays showed poor sensitivity compared to rRT-PCR (33.2% and 36.0%). rRT-PCR SARS-CoV-2 detection in self-collected GL samples had a similar PPA and NPA to that of NPSs. GL self-sampling offers a suitable and more comfortable alternative for SARS-CoV-2 detection.
- Keywords
- PCR, SARS-CoV-2, antigen assay, gargle lavage, non-invasive, self-sampling,
- MeSH
- COVID-19 * diagnosis MeSH
- Therapeutic Irrigation MeSH
- Humans MeSH
- Nasopharynx MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- SARS-CoV-2 * genetics MeSH
- Sensitivity and Specificity MeSH
- COVID-19 Testing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Where microbes colonizing skin surface may help maintain organism homeostasis, those that invade living skin layers cause disease. In bats, white-nose syndrome is a fungal skin infection that affects animals during hibernation and may lead to mortality in severe cases. Here, we inferred the amount of fungus that had invaded skin tissue of diseased animals. We used simulations to estimate the unobserved disease severity in a non-lethal wing punch biopsy and to relate the simulated pathology to the measured fungal load in paired biopsies. We found that a single white-nose syndrome skin lesion packed with spores and hyphae of the causative agent, Pseudogymnoascus destructans, contains 48.93 pg of the pathogen DNA, which amounts to about 1560 P destructans genomes in one skin lesion. Relating the information to the known UV fluorescence in Nearctic and Palearctic bats shows that Nearctic bats carry about 1.7 µg of fungal DNA per cm2, whereas Palearctic bats have 0.04 µg cm-2 of P. destructans DNA. With the information on the fungal load that had invaded the host skin, the researchers can now calculate disease severity as a function of invasive fungal growth using non-destructive UV light transillumination of each bat's wing membranes. Our results will enable and promote thorough disease severity assessment in protected bat species without the need for extensive animal and laboratory labor sacrifices.
- Keywords
- Bat, Fungal infection, Pathogen load, Pseudogymnoascus destructans, Skin lesion, UV light diagnostics, White-nose syndrome,
- MeSH
- Ascomycota * metabolism pathogenicity MeSH
- Chiroptera microbiology MeSH
- Dermatomycoses * microbiology prevention & control therapy veterinary MeSH
- Hibernation * MeSH
- Wings, Animal microbiology MeSH
- Skin microbiology MeSH
- Ultraviolet Rays * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The lack of an appropriate methodology makes numerous important issues related to miniature paintings unresolved-despite the fact that the portrait miniatures of the seventeenth to the nineteenth century represent a highly specific and significant field of European fine art. One of these issues is represented by chemical degradation of miniatures and its analytical evidence. Fragility, variability of the employed materials, and detailed execution make their analysis highly challenging-since no sampling is usually allowed and any change on their surface is immediately noticeable. Therefore, this study focused on finding a fully non-invasive multi-analytical approach to describe degradation processes resulting from the interaction of lead pigments and oils. For this purpose, a representative set of miniature portraits on various supports (ivory, metal, glass) has been selected. For the first time, Pb carboxylates (lead soaps) have been evidenced in miniatures painted in oil and also in a combined technique (gum + oil). Their distribution and crystallinity was described by a combination of X-ray-based (X-ray fluorescence and X-ray diffraction) and micro-spectroscopic methods. At the same time, a number of new findings about the employed painting technique and involvement of various pigments in the degradation processes were obtained. For example, a possible saponification of a mixed Pb-Sn-Sb yellow was indicated for the first time. Although the degradation is clearly at an advanced stage, it has not shown yet any visible symptoms that might warn restorers and curators. Therefore, without targeted analysis, it would remain overlooked.
- Keywords
- Metal soaps, Miniature paintings, Non-invasive analysis, Spectroscopy, X-ray diffraction, X-ray fluorescence,
- Publication type
- Journal Article MeSH
Reports on non-invasive blood sampling are limited, and there are only a few studies on using kissing bugs (Reduviidae) and medicinal leeches (Hirudo medicinalis) for hematology and biochemistry testing in various zoo animal species. The aim of this study was to evaluate the usefulness of non-invasive blood sampling with medicinal leeches for arbovirus epidemiological investigations in various animal species from one zoo collection. Medicinal leeches were manually applied on 35 animals of 11 species. Control blood samples were obtained by venipuncture of the jugular vein. Antibodies to tick-borne encephalitic virus (TBEV) were detected by using the immunoenzymatic method or an immunofluorescent assay (IFAT), depending on the animal species. One of the 35 animals (2.9%) was seropositive (Ovis aries), whereas the rest of the samples were seronegative in both methods of sampling (non-invasive by leeches vs. invasive by venipuncture). Blood sampling using medicinal leeches showed promising results. It is likely a good alternative to other more complex and invasive methods, and it can provide significant advancement in blood sampling for preventive medicine and epidemiological studies in zoo animals.
- Keywords
- animal welfare, epidemiology, infectious disease monitoring,
- Publication type
- Journal Article MeSH
At present, the assessment of pig welfare quality has gained significant importance, prompting the exploration of novel biomarkers for this purpose. Traditionally, these biomarkers have been monitored in the blood; however, blood sampling is considered an invasive procedure. Currently, non-invasive methods for collecting samples are emerging as viable alternatives for assessing these biomarkers. This article aims to present the current knowledge regarding the use of non-invasive methods for analysing pig welfare biomarkers, specifically focusing on the saliva, hair, faeces, and urine as matrices to determine these biomarkers. The saliva analysis encompasses various biomarkers, such as cortisol, alpha-amylase, chromogranin A, the total esterase, oxytocin, acute phase proteins, adenosine deaminase, immunoglobulins and parameters of redox homeostasis. Cortisol, a specific biomarker, can be determined in the hair, urine and faeces, while urine samples allow for the analysis of catecholamines as non-invasive markers of pig welfare.
- Keywords
- glucocorticoids, health, housing conditions, pig breeding, sow,
- Publication type
- Journal Article MeSH
- Review MeSH
Background: In an earlier monocentric study, we have developed a novel non-invasive test system for the prediction of renal allograft rejection, based on the detection of a specific urine metabolite constellation. To further validate our results in a large real-world patient cohort, we designed a multicentric observational prospective study (PARASOL) including six independent European transplant centers. This article describes the study protocol and characteristics of recruited better patients as subjects. Methods: Within the PARASOL study, urine samples were taken from renal transplant recipients when kidney biopsies were performed. According to the Banff classification, urine samples were assigned to a case group (renal allograft rejection), a control group (normal renal histology), or an additional group (kidney damage other than rejection). Results: Between June 2017 and March 2020, 972 transplant recipients were included in the trial (1,230 urine samples and matched biopsies, respectively). Overall, 237 samples (19.3%) were assigned to the case group, 541 (44.0%) to the control group, and 452 (36.7%) samples to the additional group. About 65.9% were obtained from male patients, the mean age of transplant recipients participating in the study was 53.7 ± 13.8 years. The most frequently used immunosuppressive drugs were tacrolimus (92.8%), mycophenolate mofetil (88.0%), and steroids (79.3%). Antihypertensives and antidiabetics were used in 88.0 and 27.4% of the patients, respectively. Approximately 20.9% of patients showed the presence of circulating donor-specific anti-HLA IgG antibodies at time of biopsy. Most of the samples (51.1%) were collected within the first 6 months after transplantation, 48.0% were protocol biopsies, followed by event-driven (43.6%), and follow-up biopsies (8.5%). Over time the proportion of biopsies classified into the categories Banff 4 (T-cell-mediated rejection [TCMR]) and Banff 1 (normal tissue) decreased whereas Banff 2 (antibody-mediated rejection [ABMR]) and Banff 5I (mild interstitial fibrosis and tubular atrophy) increased to 84.2 and 74.5%, respectively, after 4 years post transplantation. Patients with rejection showed worse kidney function than patients without rejection. Conclusion: The clinical characteristics of subjects recruited indicate a patient cohort typical for routine renal transplantation all over Europe. A typical shift from T-cellular early rejections episodes to later antibody mediated allograft damage over time after renal transplantation further strengthens the usefulness of our cohort for the evaluation of novel biomarkers for allograft damage.
- Keywords
- NMR-spectroscopy, biomarker, kidney transplant rejection, non-invasive test, urinary metabolites,
- Publication type
- Journal Article MeSH
Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.
No study has systematically compared the suitability of DNA methylation (DNAme) profiles in non-invasive samples for the detection of breast cancer (BC). We assess non-tumour DNAme in 1,100 cervical, buccal, and blood samples from BC cases and controls and find that cervical samples exhibit the largest nuber of differentially methylated sites, followed by buccal samples. No sites were significant in blood after FDR adjustment. Deriving DNAme-based classifiers for BC detection in each sample type (WID-buccal-, cervical-, or blood-BC), we achieve validation AUCs of 0.75, 0.66, and 0.51, respectively. Buccal and cervical BC-associated DNAme alterations distinguish between BC cases and controls in both surrogate and breast tissue (AUC > 0.88), yet individual sites and the directionality of methylation changes are not identical between these two sample types, and buccal sample DNAme aligns with breast methylation changes more closely. Pending additional validation, these insights may have the potential to improve non-invasive personalized BC prevention.
- MeSH
- Adult MeSH
- Epigenesis, Genetic * MeSH
- Epigenomics * methods MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA Methylation * MeSH
- Breast Neoplasms * genetics diagnosis MeSH
- Case-Control Studies MeSH
- Mouth Mucosa metabolism MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
