Prevalence of SARS-CoV-2 in domestic cats (Felis catus) during COVID-19 pandemic in Latvia.

BACKGROUND: The causative agent of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is of zoonotic origin and has shown reverse zoonotic transmissibility. OBJECTIVES: The aim of this cross-sectional study was to investigate the serological and molecular prevalence of SARS-CoV-2 infection in the domestic cat (Felis catus) population from Latvia in natural conditions and subsequently perform viral genome analysis. METHODS: Oropharyngeal and rectal swabs and blood samples were collected from 273 domestic cats during the second wave of COVID-19 infection in Latvia. Molecular prevalence was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR). Serum samples were analysed via double antigen enzyme-linked immunosorbent assay targeting the antibody against the nucleocapsid protein of SARS-CoV-2. Positive swab samples were analysed using whole viral genome sequencing and subsequent phylogenetic analysis of the whole genome sequencing data of the samples was performed. RESULTS: The overall SARS-CoV-2 RT-PCR positivity and seroprevalence was 1.1% (3/273) and 2.6% (7/273), respectively. The SARS-CoV-2 genome sequences from three RT-PCR positive cats were assigned to the three common lineages (PANGOLIN lineage S.1.; B.1.177.60. and B.1.1.7.) circulating in Latvia during the particular period of time. CONCLUSIONS: These findings indicate that feline infection with SARS-CoV-2 occurred during the second wave of the COVID-19 pandemic in Latvia, yet the overall prevalence was low. In addition, it seems like no special 'cat' pre-adaptations were necessary for successful infection of cats by the common lineages of SARS-CoV-2.

Evaluation of Genetic Diversity and Virulence Potential of Legionella pneumophila Isolated from Water Supply Systems of Residential Buildings in Latvia.

Legionella is an opportunistic pathogen with a biphasic life cycle that occasionally infects humans. The aim of the study was to assess the distribution of virulence genes and genetic diversity among L. pneumophila isolated from water supply systems of residential buildings in Latvia. In total, 492 water samples from 200 residential buildings were collected. Identification of Legionella spp. was performed according to ISO 11731, and 58 isolates were subjected to whole-genome sequencing. At least one Legionella-positive sample was found in 112 out of 200 apartment buildings (56.0%). The study revealed extensive sequence-type diversity, where 58 L. pneumophila isolates fell into 36 different sequence types. A total of 420 virulence genes were identified, of which 260 genes were found in all sequenced L. pneumophila isolates. The virulence genes enhC, htpB, omp28, and mip were detected in all isolates, suggesting that adhesion, attachment, and entry into host cells are enabled for all isolates. The relative frequency of virulence genes among L. pneumophila isolates was high. The high prevalence, extensive genetic diversity, and the wide range of virulence genes indicated that the virulence potential of environmental Legionella is high, and proper risk management is of key importance to public health.

Prevalence and Genetic Diversity of Legionella spp. in Hotel Water-Supply Systems in Latvia.

Legionella is one of the most important waterborne pathogens that can lead to both outbreaks and sporadic cases. The majority of travel-associated Legionnaires' disease (TALD) cases are contracted during hotel stays. The aim of this study was to evaluate the prevalence and genetic diversity of Legionella spp. in hotel water supply systems in Latvia. In total, 834 hot water samples were collected from the water systems of 80 hotels in Latvia. At least one Legionella spp. positive sample was detected in 47 out of 80 hotels (58.8%). Overall, 235 out of 834 samples (28.2%) were Legionella spp. positive. The average hot water temperature in Latvian hotels was 49.8 °C. The most predominant L. pneumophila serogroup (SG) was SG3 which was found in 113 (49.8%) positive samples from 27 hotels. For 79 sequenced L. pneumophila isolates, 21 different sequence types (ST) were obtained, including 3 new types-ST2582, ST2579, and ST2580. High Legionella contamination and high genetic diversity were found in the hotel water supply systems in Latvia, which, together with the insufficient hot water temperature, may indicate that the lack of regulation and control measures may promote the proliferation of Legionella.

Prevalence, virulence determinants, and genetic diversity in Yersinia enterocolitica isolated from slaughtered pigs and pig carcasses.

Yersinia enterocolitica is an important zoonotic foodborne pathogen that could be transferred from infected pigs to their carcasses at slaughter, with subsequent introduction of the pathogen into the food chain. The aim of the present study was to study the prevalence, virulence characteristics, and genetic diversity of Y. enterocolitica isolates present in slaughtered pig tonsils and carcasses by using the WGS approach. A total of 200 slaughtered pig tonsils from 11 pig farms were collected in 2020-2021 at six slaughterhouses located in Latvia. Out of these samples, n = 190 were obtained from slaughtered pigs raised on Latvian farms while n = 10 were of Lithuanian origin, with the number of farms sampled being 10 and 1, respectively. Additionally, 30 pig carcasses were sampled at five slaughterhouses from pigs originating from five farms in 2021. Samples were investigated microbiologically, Y. enterocolitica isolates were biotyped and serotyped. Y. enterocolitica 4/O:3 was screened for antimicrobial resistance with the EUVSEC test panels. Whole genome sequence analysis (WGS) was performed in order to detect virulence genes and to assess the genetic diversity of Y. enterocolitica isolates. A total of 139 isolates, including one to three isolates from 84 Y. enterocolitica positive slaughtered pig tonsils and 13 pig carcass samples, were subjected to WGS analysis. The prevalence of Y. enterocolitica 4/O:3 in slaughtered pig tonsils and carcasses was 35% (70/200) and 13% (4/30), respectively. Antimicrobial resistance to ampicillin and tetracycline was detected in 97% (72/74) and 1% (1/74) of Y. enterocolitica 4/O:3 isolates. Y. enterocolitica 4/O:3 was represented only by ST18, while Y. enterocolitica 1A by ST3, ST147, ST304, ST307, and ST473. The ST18 isolates harbored the same main chromosomal (ail, inv, myfA, ystA) and majority shared plasmid-borne virulence genes (virF, yadA, yop virulon). The main virulence genes were not identified within the STs of Y. enterocolitica 1A and only minor differences were found between ST3, ST147, ST304, ST307, and ST473. Among ST18 isolates, cgMLST analysis revealed 43 cgMLST genotypes while 16 cgMLST genotypes were found among Y. enterocolitica 1A STs. The present study has shown the distribution of genetically distant cgMLST genotypes in slaughtered pigs from pig farms located in different geographical regions of Latvia, with one to 11 cgMLSTs identified within each sampled farm. The presence of undistinguishable cgMLST genotypes in slaughtered pig tonsils and the respective carcasses supported the link between the slaughter of Y. enterocolitica - positive pigs and carcass contamination with Y. enterocolitica 4/O:3.

Could Artificial Intelligence/Machine Learning and Inclusion of Diet-Gut Microbiome Interactions Improve Disease Risk Prediction? Case Study: Coronary Artery Disease.

Coronary artery disease (CAD) is the most common cardiovascular disease (CVD) and the main leading cause of morbidity and mortality worldwide, posing a huge socio-economic burden to the society and health systems. Therefore, timely and precise identification of people at high risk of CAD is urgently required. Most current CAD risk prediction approaches are based on a small number of traditional risk factors (age, sex, diabetes, LDL and HDL cholesterol, smoking, systolic blood pressure) and are incompletely predictive across all patient groups, as CAD is a multi-factorial disease with complex etiology, considered to be driven by both genetic, as well as numerous environmental/lifestyle factors. Diet is one of the modifiable factors for improving lifestyle and disease prevention. However, the current rise in obesity, type 2 diabetes (T2D) and CVD/CAD indicates that the "one-size-fits-all" approach may not be efficient, due to significant variation in inter-individual responses. Recently, the gut microbiome has emerged as a potential and previously under-explored contributor to these variations. Hence, efficient integration of dietary and gut microbiome information alongside with genetic variations and clinical data holds a great promise to improve CAD risk prediction. Nevertheless, the highly complex nature of meals combined with the huge inter-individual variability of the gut microbiome poses several Big Data analytics challenges in modeling diet-gut microbiota interactions and integrating these within CAD risk prediction approaches for the development of personalized decision support systems (DSS). In this regard, the recent re-emergence of Artificial Intelligence (AI) / Machine Learning (ML) is opening intriguing perspectives, as these approaches are able to capture large and complex matrices of data, incorporating their interactions and identifying both linear and non-linear relationships. In this Mini-Review, we consider (1) the most used AI/ML approaches and their different use cases for CAD risk prediction (2) modeling of the content, choice and impact of dietary factors on CAD risk; (3) classification of individuals by their gut microbiome composition into CAD cases vs. controls and (4) modeling of the diet-gut microbiome interactions and their impact on CAD risk. Finally, we provide an outlook for putting it all together for improved CAD risk predictions.

Virulence Determinants and Genetic Diversity of Yersinia Species Isolated from Retail Meat.

Yersinia enterocolitica is an important foodborne pathogen, and the determination of its virulence factors and genetic diversity within the food chain could help understand the epidemiology of yersiniosis. The aim of the present study was to detect the prevalence, and characterize the virulence determinants and genetic diversity, of Yersinia species isolated from meat. A total of 330 samples of retailed beef (n = 150) and pork (n = 180) in Latvia were investigated with culture and molecular methods. Whole genome sequencing (WGS) was applied for the detection of virulence and genetic diversity. The antimicrobial resistance of pathogenic Y. enterocolitica isolates was detected in accordance with EUCAST. Yersinia species were isolated from 24% (79/330) of meats, and the prevalence of Y. enterocolitica in pork (24%, 44/180) was significantly higher (p < 0.05) than in beef (13%, 19/150). Y. enterocolitica pathogenic bioserovars 2/O:9 and 4/O:3 were isolated from pork samples (3%, 6/180). Only resistance to ampicillin was confirmed in Y. enterocolitica 4/O:3 and 2/O:9 isolates, but not in other antimicrobials. Major virulence determinants, including ail, inv, virF, ystA and myfA, were confirmed with WGS in Y. enterocolitica 2/O:9 and 4/O:3. MLST typing revealed 15 STs (sequence types) of Y. enterocolitica with ST12 and ST18, which were associated with pathogenic bioserovars. For Y. enterocolitica 1A, Y. kristensenii, Y. intermedia and Y. frederiksenii, novel STs were registered (ST680-688). The presence of virulence genes and genetic characteristics of certain Y. enterocolitica STs confirm the common knowledge that pork could be an important source of pathogenic Yersinia.

Determination of Fungi and Multi-Class Mycotoxins in Camelia Sinensis and Herbal Teas and Dietary Exposure Assessment.

In this paper, a study of fungal and multi-mycotoxin contamination in 140 Camellia sinensis and 26 herbal teas marketed in Latvia is discussed. The analysis was performed using two-dimensional liquid chromatography with time-of-flight mass spectrometry (2D-LC-TOF-MS) and MALDI-TOF-MS. In total, 87% of the tea samples tested positive for 32 fungal species belonging to 17 genera, with the total enumeration of moulds ranging between 1.00 × 101 and 9.00 × 104 CFU g-1. Moreover, 42% of the teas (n = 70) were contaminated by 1 to 16 mycotoxins, and 37% of these samples were positive for aflatoxins at concentrations ranging between 0.22 and 41.7 µg kg-1. Deoxynivalenol (DON) and its derivatives co-occurred in 63% of the tea samples, with their summary concentrations reaching 81.1 to 17,360 µg kg-1. Ochratoxin A (OTA), enniatins, and two Alternaria toxins were found in 10-37% of the teas at low concentrations. The dietary exposure assessment based on the assumption of a probable full transfer of determined mycotoxins into infusions indicated that the analysed teas are safe for consumers: the probable maximum daily exposure levels to OTA and the combined DON mycotoxins were only 0.88 to 2.05% and 2.50 to 78.9% of the tolerable daily intake levels.

Determination of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis by MALDI-TOF MS in clinical isolates from Latvia.

Rapid identification of methicillin-resistant Staphylococcus could ensure appropriate medical care. A total of 409 Staphylococcus spp. strains were used to develop a reliable MALDI-TOF method for species identification. We tested twelve S. aureus strains to compare three different sample preparation methods and the reproducibility of the methicillin-resistant m/z 2414 ± 2 indicator peak with direct method in triplicate. A total of 65 Staphylococcus spp. strains (including 37 methicillin-resistant strains) from clinical and hospital environment isolates were used to confirm the presence of phenol-soluble modulin (PSM-mec) peptide. All 272 S. aureus strains from 409 samples were correctly identified at species level by MALDI-TOF. The samples prepared by three methods gave spectra with differences in the intensities and presence of certain peaks. The PSM-mec peak was not visible after the extraction method. The peak m/z 2414 ± 2 was only detected in 61% of the methicillin-resistant strains and in none of the methicillin-sensitive strains. The peak reproducibility for the five analyzed S. aureus strains showing the peak at m/z 2414 ± 2 was 87%. The delta-toxin was observed in 49 out of 65 samples regardless of methicillin susceptibility, as well as in all the samples exhibiting the PSM-mec peak. The peak m/z 2414 ± 2 is specific to methicillin-resistant strains carrying the mecA gene, but the absence of peak m/z 2414 ± 2 does not exclude the possibility of resistance to methicillin. Thus, implementing MALDI-TOF analysis in routine laboratory work, especially with clinical samples, would in many cases provide rapid warning about the presence of methicillin-resistant strains.

Co-Occurrence of Free-Living Amoeba and Legionella in Drinking Water Supply Systems.

Background and Objectives: Legionella is one of the most important water-related pathogens. Inside the water supply systems and the biofilms, Legionella interact with other bacteria and free-living amoeba (FLA). Several amoebas may serve as hosts for bacteria in aquatic systems. This study aimed to investigate the co-occurrence of Legionella spp. and FLA in drinking water supply systems. Materials and Methods: A total of 268 water samples were collected from apartment buildings, hotels, and public buildings. Detection of Legionella spp. was performed in accordance with ISO 11731:2017 standard. Three different polymerase chain reaction (PCR) protocols were used to identify FLA. Results: Occurrence of Legionella varied from an average of 12.5% in cold water samples with the most frequent occurrence observed in hot water, in areas receiving untreated groundwater, where 54.0% of the samples were Legionella positive. The occurrence of FLA was significantly higher. On average, 77.2% of samples contained at least one genus of FLA and, depending on the type of sample, the occurrence of FLA could reach 95%. In the samples collected during the study, Legionella was always isolated along with FLA, no samples containing Legionella in the absence of FLA were observed. Conclusions: The data obtained in our study can help to focus on the extensive distribution, close interaction, and long-term persistence of Legionella and FLA. Lack of Legionella risk management plans and control procedures may promote further spread of Legionella in water supply systems. In addition, the high incidence of Legionella-related FLA suggests that traditional monitoring methods may not be sufficient for Legionella control.

Potential risk evaluation for unintended entry of genetically modified plant Propagating material in Europe through import of seeds and animal feed - the experience of Latvia.

Significant attention has been drawn to the adventitious and technically unavoidable presence of genetically modified (GM) organisms in the food and feed imported into the European Union (EU), while the potential presence of GM seeds in material for cultivation is less studied. Here we report a study from an EU member state, Latvia, during years 2017-2018 regarding monitoring for the presence of GM seeds in certified seed and animal feed material. Eighty-two and 28 samples of seeds intended for cultivation were analyzed in 2017 and 2018, respectively. One soybean sample contained MON40-3-2 soybean seeds (0.09 ± 0.01%) and one maize sample contained MON810 maize seeds (0.08 ± 0.01%). In addition, 102 samples of feed imported from outside of the EU or produced locally were also analyzed for the presence of genetically modified organisms (GMOs) and viability of grains. One oilseed rape cake sample contained GT73 (1.04 ± 0.01%) and one soybean cake sample contained MON40-3-2 (<0.045%). One sample of declared MON40-3-2 GM soybean cake was confirmed to be positive, with MON40-3-2 content of 94.78 ± 10.01%. One soybean sample submitted by feed producer and originating from Argentina contained 54.9 ± 1.1% of MON40-3-2 and one rapeseed sample originating from Ukraine contained 5.30 ± 3.95% of GT73. Although only two seed samples contained low levels of GMOs authorized in the EU for food and feed uses, this study reinforced the need to maintain regular monitoring programs that assist farmers in their efforts to comply with the current EU GMO legislation.

Genetically modified seeds and plant propagating material in Europe: potential routes of entrance and current status.

Genetically modified organisms (GMO), mainly crop plants, are increasingly grown worldwide leading to large trade volumes of living seeds and other plant material both for cultivation and for food and animal feed. Even though all the traded GMOs have been assessed for their safety with regards to human and animal health and the environment, there still are some concerns regarding the potential uncontrolled release in the environment of authorized or unauthorized GM plants. In this review, we identify the possible entrance routes of GM seeds and other propagating plant material in the EU which could be linked to unauthorized release of GMOs in the environment. In addition, we discuss the situation with GM plant cultivation in some non-EU countries in terms of potential risks for GM seed imports. The available body of information suggests that GM seeds and plant propagating material can enter the EU due to problems with labeling/traceability of GM seed lots, contamination of conventional seed lots and accidental release into the environment of grains imported for food and animal feed. Even though cases of uncontrolled release of authorized GMOs, as well as, release of unauthorized GMOs have been reported, they can be generally attributed to adventitious and technically unavoidable presence with little environmental impact. In conclusion, the probability of GM seeds and plant propagating material illegally entering the cultivation in EU is unlikely. However, specific monitoring programs need to be established and maintained to facilitate the compliance of European farmers with the current GMO legislation.

DNA extraction from amoebal isolates and genotype determination of Acanthamoeba from tap water in Latvia.

Acanthamoeba are free-living amoebae found in various ecological environments, and they are major microbial predators. Some Acanthamoeba species can cause keratitis, granulomatous amoebic encephalitis, and infections of the organs. This is the first study in Latvia describing Acanthamoeba detection by molecular techniques and genotyping using tap water from different sources. An 18S rRNA gene investigation of the 20 Acanthamoeba isolates suggested that 19 belonged to the hazardous T4 genotype and one sample was T12. The results indicate that tap water in Latvia may be a source of Acanthamoeba that is potentially pathogenic to humans. All obtained sequences were submitted to the GenBank database under the following accession numbers: MG386295-MG386314.

Legionella pneumophila Seropositivity-Associated Factors in Latvian Blood Donors.

Continuous environmental exposure of humans to Legionella may induce immune responses and generation of antibodies. The aim of this study was to investigate the seroprevalence of Legionella pneumophila serogroups (SG) 1-6 in the general healthy population and identify the associated host-related and environmental risk factors. L. pneumophila SG 1-6 seroprevalence among a total of 2007 blood samples collected from healthy donors was 4.8%. Seroprevalence was higher in women (5.9%) than men (3.3%) and in areas with a larger number of inhabitants, ranging from 3.5% in rural regions to 6.8% in the capital, Riga. Blood samples from inhabitants of apartment buildings tested positive for L. pneumophila in more cases (5.8%) compared to those from inhabitants of single-family homes (2.7%). Residents of buildings with a municipal hot water supply system were more likely to be seropositive for L. pneumophila (OR = 3.16, 95% CI 1.26-7.91). Previous episodes of fever were additionally identified as a risk factor (OR = 2.42, 95% CI 1.43-4.1). In conclusion, centralized hot water supply, female gender and previous episodes of fever were determined as the main factors associated with L. pneumophila seropositivity in our study population.

Prevalence of Foodborne Pathogens in Freshwater Fish in Latvia.

The aim of this study was to detect the prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish in Latvia. In total, 235 samples, including freshly caught fish from fives lakes (n = 129) and fish from retail markets (n = 106), were collected from April 2014 to December 2014 in Latvia. Samples were tested according to International Organization for Standardization methods. No Salmonella spp. were found in fresh fish from lakes or in commercially available fish. In contrast, the overall prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish was 13% (30 of 235) and 14% (34 of 235), respectively, and no significant difference between the prevalence of L. monocytogenes and Y. enterocolitica was observed (P > 0.05). All Y. enterocolitica isolates belonged to the nonpathogenic 1A biotype. Molecular serotyping of L. monocytogenes revealed that the most distributed serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2b-3b (5%), and 4b, 4d, 4e (5%). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater lake fish was 2% (2 of 129) and 3% (4 of 129), respectively. In contrast, the prevalence of L. monocytogenes and Y. enterocolitica in fish at retail markets was 26% (28 of 106) and 28% (30 of 106), respectively. In retail samples, 9 of 58 positive fish contained both L. monocytogenes and Y. enterocolitica. In general, differences in the prevalences of L. monocytogenes and Y. enterocolitica in retail samples were significantly higher than those in freshly caught fish (P < 0.05). The results of this study indicate that freshwater fish could be an important source of Y. enterocolitica and L. monocytogenes for consumers in Latvia.