Haemotropic Mycoplasma species in pet cats in Latvia : a study, phylogenetic analysis and clinical case report

Funding Information: This study was supported by ERDF project number 1.1.1.1/16/A/044. Funding Information: The authors thank Dr Liene Liga Dindonis and colleagues from Dz?vnieku Vesel?bas Centrs, R?ga, and veterinary clinic ?Vinni?, Liep?ja, for their continuous inspiration and help with collecting and managing the blood samples. Positive Mycoplasma species controls were kindly donated by Claudia Thiel, Institute of Comparative Tropical Medicine and Parasitology, University of Munich, Germany. This study was supported by ERDF project number 1.1.1.1/16/A/044. Publisher Copyright: © The Author(s) 2021.Objectives: The aim of this study was to evaluate whether haemotropic Mycoplasma species are detected in pet cats in Latvia, to perform a phylogenetic analysis of the detected pathogens and to report a clinical case of feline infectious anaemia. Methods: Peripheral blood samples (n = 125) from pet cats were submitted; 99 samples were adequate to test for the presence of Mycoplasma species DNA by nested PCR. A clinical case was added in the later stages of the study. Positive isolates were subjected to phylogenetic analysis. Results: The prevalence of ‘Candidatus Mycoplasma haemominutum’ was 15% (n = 15/99), that of Mycoplasma haemofelis was 5% (5/99) and that of ‘Candidatus Mycoplasma turicensis’ was 2% (n = 2/99). Cases of coinfection included ‘Candidatus M haemominutum’ + M haemofelis (4%; n = 4/99) and ‘Candidatus M haemominutum’ + ‘Candidatus M turicensis’ (1%; n = 1/99). This is the first published report of M haemofelis infection in the Baltic states. Two different ‘Candidatus M turicensis’ isolates were discovered after phylogenetic analysis. Conclusions and relevance: This report is the first of an autochthonous feline infectious anaemia case in the Baltic region. The prevalence of Mycoplasma species was similar to that in other northern European countries. Phylogenetic analysis revealed variability of the isolates; one of the ‘Candidatus M turicensis’ genotypes was detected for the first time in Europe.publishersversionPeer reviewe

Fatal Babesia canis canis infection in a splenectomized Estonian dog

A previously splenectomized dog from Estonia was presented with a sudden lack of appetite and discoloration of the urine. Despite supportive therapy, its condition deteriorated dramatically during 1 day. Severe thrombocytopenia and high numbers of protozoan hemoparasites were evident in blood smears, and the hematocrit dropped from 46 to 33 %. The dog was euthanized before specific antibabesial treatment was initiated. Blood samples from the dog and from two other dogs in the same household tested positive for Babesia using molecular methods, and the sequences of partial 18S rRNA gene confirmed the causative species as Babesia canis canis. The risk of severe, rapidly progressing babesiosis in splenectomized dogs merits awareness.Peer reviewe

Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis

Funding Information: This work was supported by the ERDF project no. 1.1.1.1/16/A/044. The funders of the study had no role in the study design, analysis or writing of the report. Publisher Copyright: © 2021, The Author(s).Background: Canine babesiosis and anaplasmosis, caused by Babesia canis and Anaplasma phagocytophilum, respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of B. canis and A. phagocytophilum in canine clinical samples. Methods: Sequence-based polymorphism analysis of genes encoding B. canis-specific merozoite surface protein Bc28.1 (Bc28.1) and A. phagocytophilum malate dehydrogenase (mdh) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay. Results: The presence of three B. canis Bc28.1 gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of A. phagocytophilum mdh genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for B. canis and A. phagocytophilum. The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens. Conclusions: A novel duplex RT-PCR molecular method was developed for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence variability of Bc28.1 and mdh genes indicated the genetic variability of B. canis and A. phagocytophilum isolates occurring in Latvian domestic dogs. Graphic Abstract: [Figure not available: see fulltext.].publishersversionPeer reviewe

The postmedieval Latvian oral microbiome in the context of modern dental calculus and modern dental plaque microbial profiles

Funding Information: Funding: This work was supported by ERDF grant number 1.1.1.1/16/A/101 (to R.R. and G.G.). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Recent advantages in paleomicrobiology have provided an opportunity to investigate the composition of ancient microbial ecologies. Here, using metagenome analysis, we investigated the microbial profiles of historic dental calculus retrieved from archaeological human remains from postmedieval Latvia dated 16–17th century AD and examined the associations of oral taxa and microbial diversity with specific characteristics. We evaluated the preservation of human oral micro-biome patterns in historic samples and compared the microbial composition of historic dental cal-culus, modern human dental plaque, modern human dental calculus samples and burial soil micro-biota. Overall, the results showed that the majority of microbial DNA in historic dental calculus originated from the oral microbiome with little impact of the burial environment. Good preservation of ancient DNA in historical dental calculus samples has provided reliable insight into the composition of the oral microbiome of postmedieval Latvian individuals. The relative stability of the classifiable oral microbiome composition was observed. Significant differences between the micro-biome profiles of dental calculus and dental plaque samples were identified, suggesting microbial adaptation to a specific human body environment.publishersversionPeer reviewe

Morphological characterisation and molecular sex determination of human remains from the 15th-17th centuries in Latvia

Sex determination is one of the most important and initial steps in human profile identification from archaeological material. The aim of the current study was to evaluate the application of molecular approaches alongside morphological methods for sex determination in archaeological human skeletal remains. Human skeletal remains were excavated from three cemeteries: St Gertrude Old Church, Dom Square and St Peter's Church, of 15th-17th century burials in Riga, Latvia. Morphological and molecular genetic methods, including amplification of genes AMELX/Y and SRY were used to analyse seven skeletal remains. The conducted analyses of morphological features identified sex in all seven cases (two females and five males). By molecular analyses of mediaeval DNA it was possible to determine sex in five of seven (71%) samples. In all positive cases full agreement between morphological estimation and molecular genetic methods was observed. To conclude, DNA analysis can be considered for sex identification in cases with no signs of sexual dimorphism (juvenile skeletons) or partially preserved skeletons.publishersversionPeer reviewe

LC-MS/MS method for simultaneous quantification of the first-line anti-tuberculosis drugs and six primary metabolites in patient plasma : Implications for therapeutic drug monitoring

Funding Information: This study was funded by the Latvian Council of Science. Project No: lzp-2020/1-0050. Publisher Copyright: © 2021 The AuthorsThe pharmacokinetic profiling of drug substances and corresponding metabolites in the biological matrix is one of the most informative tools for the treatment efficacy assessment. Therefore, to satisfy the need for comprehensive monitoring of anti-tuberculosis drugs in human plasma, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of first-line anti-tuberculosis drugs (ethambutol, isoniazid, pyrazinamide, and rifampicin) along with their six primary metabolites. Simple single-step protein precipitation with methanol was chosen as the most convenient sample pre-treatment method. Chromatographic separation of the ten analyte mixture was achieved within 10 minutes on a reverse-phase C8 column using mobile phase gradient mode. The multiple reaction monitoring mode (MRM) was used for analyte detection and quantification in patient samples. The chosen quantification ranges fully covered expected plasma concentrations. The method exhibited acceptable selectivity; the within- and between-run accuracy ranged from 87.2 to 113.6%, but within- and between-run precision was between 1.6 and 14.9% (at the LLOQ level CV < 20%). Although the response of the isonicotinic acid varied depending on the matrix source (CV 21.8%), validation results proved that such inconsistency does not affect the accuracy and precision of results. If stored at room temperature plasma samples should be processed within 4 h after collection, temporary storage at −20 °C up to 24 h is acceptable due to stability issues of analytes. The developed method was applied for the patient sample analysis (n = 34) receiving anti-tuberculosis treatment with the first-line drugs.publishersversionPeer reviewe

Fibronectin-binding nanoparticles for intracellular targeting addressed by B. burgdorferi BBK32 protein fragments

Virus-like particles (VLPs) are created by the self-assembly of multiple copies of envelope and/or capsid proteins from many viruses, mimicking the conformation of a native virus. Such noninfectious nanostructures are mainly used as antigen-presenting platforms, especially in vaccine research; however, some of them recently were used as scaffolds in biotechnology to produce targeted nanoparticles for intracellular delivery. This study demonstrates the creation of fusion VLPs using hepatitis B core protein-based system maintaining a fibronectin-binding property from B. burgdorferi BBK32 protein, including the evidence of particles’ transmission to BHK-21 target cells via caveolae/rafts endocythosis. These results make this construct to be an attractive model in development of HBc-based nanoparticles for cellular targeting applications and highlights the fragment of B. burgdorferi BBK32 as a novel cellular uptake-promoting peptide.This work was supported by grant of Latvian Council of Science, Nr. 10.0029.3 and by ESF Projec

Development of rapid antigen test prototype for detection of SARS-CoV-2 in saliva samples

Funding Information: This study was supported by VPP Program, Republic of Latvia Ministry of Education and Science, project No. VPP-COVID-2020/1-0025. Publisher Copyright: © 2022 Taylor and Francis Ltd. All rights reserved.Background: The development of easy-to-perform diagnostic methods is highly important for detecting current coronavirus disease (COVID-19). This pilot study aimed at developing a lateral flow assay (LFA)- based test prototype to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva samples. Methods: Mice were immunized using the recombinant receptor-binding domain (rRBD) of SARS-CoV-2 virus spike protein. The combinations of the obtained mouse anti-receptor-binding domain (RBD) polyclonal antibodies (PAbs) and several commercial antibodies directed against the SARS-CoV-2 spike protein were used for enzyme-linked immunosorbent assay (ELISA) to select antibody pairs for LFA. The antibody pairs were tested in a LFA format using saliva samples from individuals with early SARS-CoV-2 infection (n = 9). The diagnostic performance of the developed LFA was evaluated using saliva samples from hospitalized COVID-19 patients (n = 111); the median time from the onset of symptoms to sample collection was 10 days (0-24 days, interquartile range (IQR): 7-13). The reverse transcription-polymerase chain reaction (rRT-PCR) was used as a reference method. Results: Based on ELISA and preliminary LFA results, a combination of mouse anti-RBD PAbs (capture antibody) and rabbit anti-spike PAbs (detection antibody) was chosen for clinical analysis of sample. When compared with rRT-PCR results, LFA exhibited 26.5% sensitivity, 58.1% specificity, 50.0% positive prediction value (PPV), 33.3% negative prediction value (NPV), and 38.7% diagnostic accuracy. However, there was a reasonable improvement in assay specificity (85.7%) and PPV (91.7%) when samples were stratified based on the sampling time. Conclusion: The developed LFA assay demonstrated a potential of SARS-CoV-2 detection in saliva samples. Further technical assay improvements should be made to enhance diagnostic performance followed by a validation study in a larger cohort of both asymptomatic and symptomatic patients in the early stage of infection.Peer reviewe

Babesiosis in Southeastern, Central and Northeastern Europe: An Emerging and Re-Emerging Tick-Borne Disease of Humans and Animals

There is now considerable evidence that in Europe, babesiosis is an emerging infectious disease, with some of the causative species spreading as a consequence of the increasing range of their tick vector hosts. In this review, we summarize both the historic records and recent findings on the occurrence and incidence of babesiosis in 20 European countries located in southeastern Europe (Bosnia and Herzegovina, Croatia, and Serbia), central Europe (Austria, the Czech Republic, Germany, Hungary, Luxembourg, Poland, Slovakia, Slovenia, and Switzerland), and northern and northeastern Europe (Lithuania, Latvia, Estonia, Iceland, Denmark, Finland, Sweden, and Norway), identified in humans and selected species of domesticated animals (cats, dogs, horses, and cattle). Recorded cases of human babesiosis are still rare, but their number is expected to rise in the coming years. This is because of the widespread and longer seasonal activity of Ixodes ricinus as a result of climate change and because of the more extensive use of better molecular diagnostic methods. Bovine babesiosis has a re-emerging potential because of the likely loss of herd immunity, while canine babesiosis is rapidly expanding in central and northeastern Europe, its occurrence correlating with the rapid, successful expansion of the ornate dog tick (Dermacentor reticulatus) populations in Europe. Taken together, our analysis of the available reports shows clear evidence of an increasing annual incidence of babesiosis across Europe in both humans and animals that is changing in line with similar increases in the incidence of other tick-borne diseases. This situation is of major concern, and we recommend more extensive and frequent, standardized monitoring using a “One Health” approach