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Various muscle samples of wild boar (Sus scrofa) from Latvia were studied for the presence of Sarcocystis infection by means of morphological and molecular methods. Sarcocysts were detected in 122 out of 140 (87.1%) wild boar examined. According to the morphological appearance of sarcocysts, the observed cysts belonged to one morphological type and resembled Sarcocystis miescheriana. Twenty-three sarcocysts isolated from the muscles of Latvian wild boars were molecularly characterized at 18S rRNA, ITS1 and cox1. Additionally, eight sarcocysts obtained from Lithuanian wild boars were subjected to molecular analysis in order to compare intraspecific genetic variability. The amplified 18S rRNA region using newly designed primers is sufficiently variable to separate S. miecheriana from S. suihominis. All Latvian and Lithuanian isolates were confirmed belonging to S. miescheriana. No genetic variation was detected within 18S rRNA and ITS1. By contrast, the high intraspecific genetic variability of S. miescheriana was observed within cox1 since each newly obtained sequence represented a unique haplotype. The comparison made using S. miescheriana isolates from Italian and Japanese wild boar and Chinese domestic pig revealed the genetic similarity of the samples depending on their geographical distances. The current study provides the first detection of Sarcocystis infection in wild boars from Latvia and molecular characterization of S. miescheriana.

We investigated factors affecting species composition and patterns of species associations in parasite communities of water frogs (Pelophylax esculentus complex), applying the distance-based redundancy analysis for component communities (assemblages harboured by host populations) and Markov random fields modelling for infracommunities (assemblages harboured by individual hosts), respectively. We asked (a) What are the relative effects of variation in environmental, land use (i.e., anthropogenic), and spatial factors on the variation in the species composition of component communities (i.e., in a locality)? and (b) What is the dominant pattern of species associations in infracommunities (in a host individual), and how do these associations vary along environmental and/or anthropogenic gradients? In component communities, the greatest portion of variation in helminth species composition was explained by the combined effects of space, anthropogenic pressure, and NDVI, with the pure effect of the spatial predictor being much stronger than the effects of the remaining predictors. In infracommunities, the probability of occurrence of some, but not all, helminth species depended on the occurrence of another species, with the numbers of negative and positive co-occurrences being equal. The strength and/or sign of associations of some species pairs were spatially stable, whereas interactions between other species pairs varied along the gradient of the amount of green vegetation, from negative to positive and vice versa. We conclude that the processes in parasite infracommunities and component communities in frogs are intertwined, with both bottom-up and top-down effects acting at different hierarchical scales.

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

Various muscle tissue samples from 60 moose (Alces alces) in the Baltic region were examined for Sarcocystis species. Sarcocysts were detected in 49 out of 60 (81.7%) moose investigated. Six species, Sarcocystis alces, Sarcocystis hjorti, Sarcocystis linearis, Sarcocystis silva, Sarcocystis ovalis, and Sarcocystis sp., were identified using light microscopy (LM), and DNA sequence analysis (cox1 and 18S rDNA). Sarcocysts of S. alces, S. ovalis, and S. hjorti were studied using transmission electron microscopy (TEM); sarcocyst walls of S. alces, S. ovalis, and S. hjorti were type 25, type 24, and type 7a, respectively. Sarcocystis linearis previously found in roe deer and red deer was also shown to use moose as an intermediate host for the first time. The unknown Sarcocystis sp. was rare and might employ another main intermediate host. Phylogenetic results demonstrated that Sarcocystis sp. was most closely related to Sarcocystis tarandivulpes, using canids as definitive hosts.

Publisher's version (útgefin grein), The world is changing, and parasites adapt. The Nordic-Baltic region in northern Europe – including the Nordic countries Denmark, Finland, Iceland, Norway and Sweden, and the Baltic States Estonia, Latvia and Lithuania – is facing new parasitological challenges due to changes in populations of parasites and their hosts and the spread of new parasites to the region due to climate change. Some changes can also be ascribed to increased awareness and detection. In this paper, we review and discuss a convenience selection of ten timely examples of recent observations that exemplify trends and challenges from different fields of parasitology, with particular focus on climate change and potential changes in epidemiology of pathogens in northern Europe. The examples illustrate how addressing parasitological challenges often requires both intersectoral and international collaboration, and how using both historical baseline data and modern methodologies are needed., The authors thank Brian Lassen for the graphical abstract. The World Federation for Parasitologists is thanked for this Special Issue initiative.

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.

Toxoplasma gondii is an important zoonotic parasite that infects a wide spectrum of mammals. Moreover, its presence in wild carnivores is indicative of environmental contamination by the parasite. A total of 186 marten (152 pine marten; 34 stone marten) meat juice samples from Latvia and Lithuania were tested for T . gondii seroprevalence. Samples were tested for T . gondii specific antibodies by a commercial ELISA and antibodies were found in 121 (65.1%) samples. Higher prevalence (67.8%) occurred in pine martens compared to that of stone martens (52.9%). Adult pine martens had a 2.0 (95% CI 0.9–4.7) times higher odds ratio to test seropositive than juvenile Pine martens. In addition, a significant positive correlation ( r = 0.75; P = 0.05) was observed between T . gondii seroprevalence in martens and the number of estimated Eurasian lynx as a possible definitive host within the game administrative unit. The present study suggests that the high seroprevalence of T . gondii in Pine and Stone martens could indicate a high incidence of the parasite in the intermediate and definitive host population and in the environment.

An increasing number of bacteria have been described as benefiting from interaction with free-living amoeba. The most common association between free-living amoeba and microorganisms is interaction of various non-pathogenic and pathogenic bacterial species with amoeba. Various pathogenic bacterial species have capacity to resist digestion by free-living amoeba, which has been observed by many researchers. Also, several of these pathogens are able to resist digestion by macrophages. In addition, free-living amoeba have been associated with several diseases in humans. Acanthamniioeba castella is an important predator of bacteria. It is a ubiquitous organism in water, soil, and air. Attention from a public health perspective is needed by investigation of interaction of foodborne pathogens and free-living amoeba. Bacteria can use free-living amoeba as reservoirs, mediators or vehicles, an infection route, “biological gym” and evolutionary crib or interaction may result in a close endosymbiotic relationship. The purpose of this review is to describe the interaction mechanisms between free-living amoeba and common bacteria species that survive in host cells.

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

Rodents have been widely studied as intermediate hosts of Sarcocystis; however, only a few reports on these parasites in the black rat (Rattus rattus) are known. Having examined 13 black rats captured in Latvia, sarcocysts were found in skeletal muscles of two mammals and were described as Sarcocystis ratti n. sp. Under a light microscope, sarcocysts were ribbon-shaped, 0.9–1.3 × 0.09–0.14 mm in size and had a thin (0.8–1.3 μm) and smooth cyst wall. The lancet-shaped bradyzoites were 8.3 × 4.3 (7.5–9.3 × 3.9–4.8) μm. Under a transmission electron microscope, the cyst wall was up to 1.3 μm thick, wavy, the ground substance appeared smooth, type 1a-like. Morphologically, sarcocysts of S. ratti were somewhat similar to those of S. cymruensis, S. rodentifelis, and S. dispersa-like previously identified in the brown rat (Rattus norvegicus). On the basis of 18S rDNA, 28S rDNA, and cox1, significant genetic differences (at least 2.3, 4.5, and 5.8%, respectively) were observed when comparing S. ratti with other Sarcocystis species using rodents as intermediate hosts. While ITS1 sequences of S. ratti were highly distinct from other Sarcocystis species available in GenBank. Phylogenetic and ecological data suggest that predatory mammals living near households are definitive hosts of S. ratti.

Luminescent derivatives of benzanthrone are becoming more useful based on their light-absorbing and fluorescent-emitting properties. Our previous studies showed that luminescent staining properties of the same benzanthrone dye differ for variable parasite samples. Therefore, two types of benzanthrone dyes were prepared. One has a strongly basic amidine group and a halogen atom, and the other has an amide moiety and a tertiary amine group. Trematoda Parafasciolopsis fasciolaemorpha is a liver fluke of a moose (Alces alces) and has a significant influence on the health and abundance of the moose population. Staining protocols for parasite P. fasciolaemorpha specific organ or organ systems imaging are mostly time-consuming and labor-intensive. The study aimed to compare the fixation technique and the staining protocol by synthesized benzanthrone luminescent dyes to determine detailed morphology, anatomical arrangement of the organ systems and gross organization of the muscle layers of P. fasciolaemorpha using confocal laser scanning microscopy. Luminophores were tested for samples fixed in different fixatives. Developed dyes and staining protocol resulting in imaging of all parts of trematode without additional sample preparation procedures, which usually are required for parasite examination. Obtained results confirmed that the most qualitative results could be reached using 3-N-(2-piperidinylacetamido)benzanthrone dye which has amide moiety and a tertiary amine group. Based on obtained results, 3-N-(2-piperidinylacetamido)benzanthrone gave more qualitative parasite visualization than 2-bromo-3-N-(N′,N′-dimethylformamidino)benzanthrone.

Parasites of the genus Trichinella are cosmopolitan nematodes infecting primarily wild animals, which represent the main reservoirs of these zoonotic pathogens. To investigate the transmission patterns of Trichinella spp. from wild to domestic animals and to humans and for the risk assessment of these parasites in a geographical area, it is important to know the number of possible transmission events deriving from carcasses of infected hosts. For this purpose, the evaluation of the larval biomass in reservoir hosts is needed. No data is available on how to estimate the biomass of Trichinella spp. larvae in muscles of naturally infected animals. The aim of this study was to evaluate the larval biomass in naturally infected pine martens (Martes martes) of Latvia, in which the prevalence of Trichinella britovi infection was over 50%. Single muscles or group of muscles (abdomen, back, diaphragm, intercostal muscles, muscles from the head, left and right shoulders, lower and upper parts of the forelimbs and hind limbs, neck, rump with tail, and base and tip of the tongue) were collected from five skinned and eviscerated carcasses of T. britovi infected pine martens. Muscles were entirely removed from the bones and weighted. Each muscle or group of muscles was separately digested to detect the larvae per gram (LPG). Using linear regression, the larval burden in each muscle or group of muscles was evaluated to measure the possible prediction of the total animal larval burden (both as total number of larvae and as average LPG). All muscles were significantly predictive of the total burden with high "goodness of fit" (all adjusted R2>0.80; P≤0.01), and the left shoulder provided the highest adjusted R2 (0.999). Then, to estimate the Trichinella britovi biomass in the pine marten population of Latvia, recent literature data on prevalence (56.2%, 95% CI: 47.8-64.3) and geometric mean LPG (1.26, 95% CI: 0.89-1.79) in the limb muscles of a sample representative of the whole Latvian pine marten population, were used. Using the predictive estimated relationship between LPG in the limb and that in the entire animals and by the estimated animal population and their mean total muscle weight, the Trichinella britovi biomass in the pine marten population of Latvia was estimated to be of 6,647,092 (95% CI: 3,840,030-11,100,000) larvae. The assessment of the biomass in nature can help to understand the epidemiological pattern of these pathogens, to implement actions aimed at controlling the infection in target animal species, and to acquire basic information on the complex biology of this group of zoonotic nematodes.

We investigated the spatial distribution and measured the hard parts of the attachment apparatuses and reproductive organs of the 2 eel-specific monogenean species Pseudodactylogyrus anguillae and Pseudodactylogyrus bini. Comparative analysis focused on parasite microhabitats within the gill apparatus. Additionally, we compared the morphometric characteristics of the monogeneans based on geographical locality and seasonality. The attachment apparatus and reproductive organ characteristics of the monogeneans were similar in all gill apparatus microhabitats. However, measurements differed significantly between localities and between seasons, with the results suggesting that abiotic factors play a larger role in determining parasite morphology than do the gill microhabitats.

Benzanthrone derivates are now widely used in many industrial and scientific applications as dyes for polymers and textiles. In biochemical, biomedical and diagnostics investigations benzanthrone dyes are used as a lipophilic fluorescent probe since many benzanthrone derivates demonstrate bright fluorescence and they have ability to intercalate between membrane lipids. The aim of research presented here was to assess the luminescence ability of benzanthrone derivatives using microscopic visualization of biological objects. Accordingly, specimens of freshwater trematodes: Diplostomum spathaceum, Diplodiscus subclavatus and Prosotocus confusus, were stained by novel benzanthrone dyes using different fixatives. The samples were examined under a confocal laser scanning microscope. All of the dyes tested demonstrated good results for digestive and reproductive system visualization. Based on obtained results we conclude that benzanthrone dyes could be used for internal and external structure confocal laser scanning microscopic imaging of trematode specimens.

Background Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Methods Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Results Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Conclusions Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

Benzanthrone derivates are now widely used in many industrial and scientific applications as dyes for polymers and textiles. In biochemical, biomedical and diagnostics investigations benzanthrone dyes are used as a lipophilic fluorescent probe since many benzanthrone derivates demonstrate bright fluorescence and they have ability to intercalate between membrane lipids. The aim of research presented here was to assess the luminescence ability of benzanthrone derivatives using microscopic visualization of biological objects. Accordingly, specimens of freshwater trematodes: Diplostomum spathaceum, Diplodiscus subclavatus and Prosotocus confusus, were stained by novel benzanthrone dyes using different fixatives. The samples were examined under a confocal laser scanning microscope. All of the dyes tested demonstrated good results for digestive and reproductive system visualization. Based on obtained results we conclude that benzanthrone dyes could be used for internal and external structure confocal laser scanning microscopic imaging of trematode specimens.

Consumption of undercooked pork products is considered a major risk factor for contracting toxoplasmosis in humans in several countries. In total, 803 pork samples and 606 wild boar meat samples were collected from different regions of Latvia during June 2010 and February 2011 were tested for Toxoplasma gondii antibodies using an in-house ELISA assay. Seroprevalence in wild boar (33.2%, P0.05) was significantly higher than in domestic pig (4.2%). The prevalence of T. gondii -specific antibodies was greater in free-ranging domestic pigs (6.2%, P0.05) than those that were intensively farmed (0.4%). Animals from free-range farms had 17.6 times higher odds ratio (OR) (95% confidence interval [CI]: 2.4-129.8) for the presence of specific T. gondii antibodies than animals from intensive farms. The OR (95% CI: 1.1-2.7) of feral wild boars for T. gondii -specific antibodies was 1.7 times higher than for farmed wild boars. Our findings indicate that the prevalence of T. gondii infection was higher for animals with open access to wildlife and direct contact with environment. This is the first report of T. gondii prevalence in pigs in Latvia.

Trichinella spp. are zoonotic parasites transmitted to humans by the consumption of raw or insufficiently cooked meat of different animal species. Carnivore mammals are important reservoir hosts of these nematodes. The aims of this work were to establish the prevalence of Trichinella spp. and infection intensity in sylvatic carnivore mammals of Latvia, to identify the etiological agents at the species level and their circulation in the Latvian regions. From 2010 to 2014, muscle samples were collected from 1286 hunted animals (2 European badgers, 137 pine martens, 24 stone martens, 4 golden jackals, 394 raccoon dogs, 668 red foxes, 23 grey wolves, and 34 Eurasian lynxes). Trichinella spp. larvae were isolated by muscle digestion. Overall, 633 animals (49.2%; 95% CI 46.5%-52.0%) belonging to all the eight investigated species, tested positive for Trichinella spp. larvae. Trichinella britovi was the most common species (94.0%; 95% CI 91.7%-95.7%). Trichinella nativa was detected in 30 animals as single (6, 1.1%; 95% CI 0.4%-2.3%) or mixed infection (24, 4.4%; 95% CI 2.9%-6.4%) with T. britovi. Trichinella spiralis was detected in only three animals as mixed infection with T. britovi. The high prevalence of Trichinella spp. infection in sylvatic carnivore mammals suggests that they are good indicators for the risk assessment of Trichinella spp. in Latvia.

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.

Felids are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that shed resistant oocysts in the environment. A total of 242 serum samples and 80 fecal samples from domestic cats in Latvia was tested for T. gondii infection. Serum samples were tested for T. gondii antibodies by an indirect in-house ELISA; antibodies were found in 125 (51.6%) of 242 cats; seroprevalence increased with age, indicating postnatal infection. Using multiple logistic regression analyses, age and outdoor access were found to be the most significant (F = 21.70, P < 0.05) factors associated with T. gondii infection in Latvia. Toxoplasma gondii-like oocysts were detected in 2 of the cats examined microscopically using the salt flotation method, but definitive diagnosis could not be made because a bioassay was not performed.

In the article, preliminary data on the helminthofauna of the Eurasian lynx (Lynx lynx) in Latvia are presented. Material for the research on the helminthofauna was collected and investigated from 1999 to 2002. A total of 42 individuals was examined for helminths. Helminth species occurring most frequently in the Eurasian lynx and helminths dangerous to people were established. Six helminth species belonging to the following taxonomic groups were found: one species (Taenia pisiformis) of Cestoda and five species (Toxocara mystax, Thominx aerophilus, Trichinella sp., Capillaria felis-cati, and Nematoda sp.) of Nematoda.

Background Trichinella spp. are zoonotic parasites transmitted to humans by the consumption of raw or insufficiently cooked meat of different animal species. The most common source of infection for humans is meat from pigs and wild boar (Sus scrofa). The aim of the present work was to evaluate the incidence of Trichinella spp. infections in wild boar hunted in Latvia over a 38 year interval (1976 to 2013). Methods A total 120,609 wild boars were individually tested for Trichinella spp. by trichinoscopy and, in case of negativity, by artificial digestion of 25 g muscles, in the 1976–2005 period, and by artificial digestion of 25–50 g muscles in the 2006–2013 period. Trichinella spp. larvae were identified at the species level by multiplex PCR. Results In the study period, the overall prevalence of infected wild boar was 2.5%. Trichinella britovi was the predominant (90%) species. The incidence of Trichinella spp. infection in wild boar exhibited two different trends. From 1976 to 1987, the incidence of infected/hunted wild boar increased from 0.23% to 2.56%, then it decreased to 0.19 in 1994. Thereafter, the incidence fluctuated between 0.05% and 0.37%. A statistically significant (P