Comparative Microbiome Analysis of Three Epidemiologically Important Tick Species in Latvia.

(1) Background: Amplicon-based 16S rRNA profiling is widely used to study whole communities of prokaryotes in many niches. Here, we comparatively examined the microbial composition of three tick species, Ixodes ricinus, Ixodes persulcatus and Dermacentor reticulatus, which were field-collected in Latvia. (2) Methods: Tick DNA samples were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). (3) Results: The results showed significant differences in microbial species diversity and composition by tick species and life stage. A close similarity between microbiomes of I. ricinus and I. persulcatus ticks was observed, while the D. reticulatus microbiome composition appeared to be more distinct. Significant differences in alpha and beta microbial diversity were observed between Ixodes tick life stages and sexes, with lower taxa richness indexes obtained for female ticks. The Francisella genus was closely associated with D. reticulatus ticks, while endosymbionts Candidatus Midichlorii and Candidatus Lariskella were associated with I. ricinus and I. persulcatus females, respectively. In I. ricinus females, the endosymbiont load negatively correlated with the presence of the Rickettsia genus. (4) Conclusions: The results of this study revealed important associations between ticks and their microbial community and highlighted the microbiome features of three tick species in Latvia.

Impact of Growth Conditions on the Viability of Trichoderma asperellum during Storage.

As excellent biocontrol agents and plant growth promoters, Trichoderma species are agriculturally important. Trichoderma spp. cultures can be produced using solid-state or submerged cultivation, the latter being much less labor intensive and easier to control and automate. The aim of the study was to investigate the ability to increase the shelf-life of T. asperellum cells by optimizing cultivation media and upscaling the submerged cultivation process. Four different cultivation media were used with or without the addition of Tween 80 and stored with or without incorporation into peat, and viability, expressed as CFU/g, was assessed during one year of storage in an industrial warehouse. The addition of Tween 80 had a positive effect on the biomass yield. The culture medium played a major role in the ability of the mycelium to produce spores, which in turn influenced the amount of CFU. This effect was less pronounced when the biomass was mixed with peat prior to storage. A procedure that increases the number of CFU in a peat-based product formulation is recommended, namely, incubation of the mixture at 30 °C for 10 days prior to storage at 15 °C over an extended period of time.

Oral microbiome variations related to ageing: possible implications beyond oral health.

The global population is getting older due to a combination of longer life expectancy and declining birth rates. Growing evidence suggests that the oral microbiota composition and distribution may have a profound effect on how well we age. The purpose of this study was to investigate age-related oral microbiome variations of supragingival plaque and buccal mucosa samples in the general population in Latvia. Our results indicated significant difference between supragingival plaque bacterial profiles of three age groups (20-40; 40-60; 60 + years). Within supragingival plaque samples, age group 20-40 showed the highest bacterial diversity with a decline during the 40-60 age period and uprise again after the age of 60. Among other differences, the important oral commensal Neisseria had declined after the age of 40. Additionally, prevalence of two well-documented opportunistic pathogens Streptococcus anginosus and Gemella sanguinis gradually rose with age within our samples. Furthermore, supragingival plaque and buccal mucosa samples significantly differed in overall bacterial composition.

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

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.

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

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.

Large-scale countrywide screening for tick-borne pathogens in field-collected ticks in Latvia during 2017-2019.

BACKGROUND: Tick-borne diseases are of substantial concern worldwide in both humans and animals. Several hard tick species are of medical and veterinary interest in Europe, and changes in the range of tick species can affect the spread of zoonotic pathogens. The aim of the present study was to map the current prevalence and distribution pattern of ticks and related tick-borne pathogens in Latvia, a Baltic state in northern Europe. METHODS: Nearly 4600 Ixodes ricinus, I. persulcatus and Dermacentor reticulatus tick samples were collected in all regions of Latvia during 2017-2019 and were screened by molecular methods to reveal the prevalence and distribution pattern of a wide spectrum of tick-borne pathogens. RESULTS: New localities of D. reticulatus occurrence were found in western and central Latvia, including the Riga region, indicating that the northern border of D. reticulatus in Europe has moved farther to the north. Among the analyzed ticks, 33.42% carried at least one tick-borne pathogen, and 5.55% of tick samples were positive for two or three pathogens. A higher overall prevalence of tick-borne pathogens was observed in I. ricinus (34.92%) and I. persulcatus (31.65%) than in D. reticulatus (24.2%). The molecular analysis revealed the presence of tick-borne encephalitis virus, Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia spp. Overall, 15 and 7 tick-borne pathogen species were detected in Ixodes spp. and D. reticulatus ticks, respectively. This is the first report of Borrelia miyamotoi in Latvian field-collected ticks. CONCLUSIONS: This large-scale countrywide study provides a snapshot of the current distribution patterns of Ixodes and Dermacentor ticks in Latvia and gives us a reliable overview of tick-borne pathogens in Latvian field-collected ticks.

Babesiosis in Latvian domestic dogs, 2016-2019.

Canine babesiosis is tick-borne infection that represents a major veterinary issue in Central and Eastern Europe with a tendency to expand northwards. The first published report in Latvia about autochthonous cases of babesiosis in domestic dogs with no travel history was in 2013, and to the best of our knowledge, no other studies on this issue have been published to date. The aim of this study was to analyze the occurrence and clinical manifestations of babesiosis in Latvian domestic dogs with a history of tick exposure to determine the extent to which Babesia sp. causes the disease and to map outbreaks in Latvia. From 2016 to 2019, blood samples from dogs were collected, and molecular testing was performed by nested PCR using Babesia sp.-specific primers. In total, 43 of 262 samples were Babesia canis-positive. A seasonal pattern was observed for the outbreaks, as the majority of B. canis-positive samples (98%) were submitted between April and June, and there was a single canine babesiosis case recorded in October. Nearly half of the cases (46.5%) were recorded in the capital, Riga, and other cases were recorded in southern and western parts of Latvia. Clinical signs were consistent with typical manifestations of acute canine babesiosis; most common hematological changes were thrombocytopenia (89%) and normocytic normochromic anemia (69%). Blood smear microscopy was positive for 79% of cases. Two B. canis genotypes were distinguished on the basis of two nucleotide (GA → AG) substitutions in the 18S rRNA gene at positions 610/611; however, no relationship between the genotypes and the severity of the disease was found. In conclusion, canine babesiosis has become an endemic disease in the southern and western regions of Latvia and is caused solely by the large babesia species B. canis. Awareness among veterinarians and pet owners regarding the disease should be increased.

Tick-borne pathogens in ticks collected from dogs, Latvia, 2011-2016.

BACKGROUND: Different tick species are able to transmit different pathogens, and tick-borne diseases are of substantial concern worldwide for both humans and animals. Environmental changes and changes in the range of tick species, including Dermacentor reticulatus in Europe, can affect the spread of zoonotic pathogens. The aim of this study was to investigate the prevalence of the tick-borne pathogens in ticks removed from dogs in Latvia, and to explore possible changes between years 2011 and 2016. RESULTS: In 2011, only Ixodes ticks (221 Ixodes ricinus and 22 Ixodes persulcatus) were collected from dogs, while in 2016 tick samples belonged to Ixodes ricinus (360), Ixodes persulcatus (2) and Dermacentor reticulatus (27) species. In total, 35.8 and 40.0% of adult ticks were pathogen-positive in 2011 and 2016, respectively; the difference was not statistically significant (P > 0.05). The molecular analysis indicated the presence of 13 tick-borne microorganisms; the most prevalent pathogen was Rickettsia, followed by Borrelia burgdorferi sensu lato group spirochetes, Anaplasma phagocytophilum and Babesia species. Borrelia miyamotoi was also present. A co-infection with two and three tick-borne pathogens was detected in 7.9 and 7.4% of Ixodes ricinus and Dermacentor reticulatus, respectively. The results of this study confirmed that the spread of novel vectors could bring additional risk of exposure to novel emerging pathogens to pets and their owners, as both Babesia canis and Rickettsia raoultii were shown to be highly associated with Dermacentor reticulatus ticks in Latvia. CONCLUSIONS: This study demonstrates the potential danger from the inadvertent introduction of novel disease pathogens and vectors. Awareness of co-infections and Dermacentor reticulatus-related pathogens needs to be increased.