Tannins Can Have Direct Interactions with Anthelmintics: Investigations by Isothermal Titration Calorimetry.

Plant tannins are known for their anthelmintic and antiparasitic activities and have been increasingly studied to battle the ever-growing problem of anthelmintic resistance. While tannins have been shown to exhibit these activities on their own, one approach would be to use them as complementary nutrients alongside commercial anthelmintics. So far, research on the interactions between tannins and anthelmintics is limited, and few studies have reported both synergistic and antagonistic effects depending on the type of tannin and the method used. These interactions could either strengthen or weaken the efficacy of commercial anthelmintics, especially if tannin-rich diets are combined with anthelmintics used as oral drenches. To study these interactions, a series of hydrolysable tannins (HTs) was selected, and their direct interactions with thiabendazole (TBZ) were evaluated by isothermal titration calorimetry (ITC), which allowed the detection of the exothermic interaction but also the roles and significances of different structural features of HTs in these interactions. Our results show that HTs can have a direct interaction with the benzimidazole anthelmintic TBZ and that the interaction is strengthened by increasing the number of free galloyl groups and the overall molecular flexibility of HTs.

Label-free quantitative proteomics and immunoblotting identifies immunoreactive and other excretory-secretory (E/S) proteins of Anoplocephala perfoliata.

Anoplocephala perfoliata is a common tapeworm in horses causing colic and even mortalities. Current diagnostic tests to detect A. perfoliata infections have their limitations and an improved method is needed. Immunoreactive excretory/secretory proteins (E/S proteome) of this parasite can provide promising candidates for diagnostic tests. We compared E/S proteins produced by small (length < 20 mm, width < 5 mm) and large (length 20 to 40 mm, width 5 to 10 mm) A. perfoliata worms in vitro by label-free quantitative proteomics using a database composed of related Hymenolepis diminuta, Echinococcus multilocularis/granulosus and Taenia aseatica proteins for protein identifications. Altogether, 509 E/S proteins were identified after incubating the worms in vitro for three and eight hours. The greatest E/S proteome changes suggested both worm size- and time-dependent changes in cytoskeleton remodeling, apoptosis, and production of antigens/immunogens. The E/S proteins collected at the three-hour time point represented the natural conditions better than those collected at the eight-hour time point, and thereby contained the most relevant diagnostic targets. Immunoblotting using antibodies from horses tested positive/negative for A. perfoliata indicated strongest antigenicity/immunogenicity with 13-, 30- and 100-kDa proteins, involving a thioredoxin, heat-shock chaperone 90 (Hsp90), dynein light chain component (DYNLL), tubulin-specific chaperone A (TBCA) and signaling pathway modulators (14-3-3 and Sj-Ts4). This is among the first studies identifying new diagnostic targets and A. perfoliata antigens eliciting a IgG-response in horses.

Dispersal of taeniid eggs: Experimental faecal contamination of forest environment followed by DNA detection in wild berries.

To understand Taeniidae epidemiology, the principles of egg-dispersion dynamics under natural conditions must be known. In this study, non-zoonotic Taenia laticollis was used as a model parasite for the family Taeniidae (including Echinococcus spp.). An experiment to investigate dispersion from contaminated faeces to the surroundings was performed both with bilberries (Vaccinium myrtillus) and lingonberries (Vaccinium vitis-idaea), both of which are commercially harvested wild berries in Finland. For this experiment, 30 g of fox faeces was inoculated with 30,000 T. laticollis eggs for the bilberry experiment and 100,000 eggs for the lingonberry experiment. The faecal material was placed in the middle of good berry growth areas in four locations for bilberries and eight locations for lingonberries. After 41-42 days, berries at different distances (0-15 m) from the original contamination spot were collected and delivered to our laboratory. DNA was extracted from washed and sieved material and analysed using T. laticollis-specific semi-quantitative SYBR Green real-time polymerase chain reaction (qPCR). Taenia laticollis-specific DNA was recovered from 67% (8/12) of bilberry samples but not reliably from any of the lingonberry samples 0% (0/24), although the exposure dose was higher for those. The qPCR results suggest that under natural conditions, taeniid egg dispersion from the contamination spot is demonstrated but attachment is berry specific. The surface of bilberries may be more adhesive for taeniid eggs than the waxier and harder pericarp of the lingonberries or there might be a difference in the dispersal mechanism caused by different biotopes.

Seroprevalence of Encephalitozoon cuniculi and Toxoplasma gondii antibodies and risk-factor assessment for Encephalitozoon cuniculi seroprevalence in Finnish pet rabbits (Oryctolagus cuniculus).

BACKGROUND: Neurological signs, such as head tilt, torticollis, paralysis, and seizures, are common in rabbits. Differential diagnoses include two zoonotic infections caused by the microsporidial fungi Encephalitozoon cuniculi and the apicomplexan protozoa Toxoplasma gondii. Both infections are mainly latent in rabbits but may cause severe or even fatal disease. Although several international studies have reported the seroprevalence of these pathogens in different commercial rabbit populations, similar prevalence studies and risk-factor analyses among family-owned pet rabbits are uncommon and lacking in Scandinavia. We sought to estimate the seroprevalence and possible risk factors for E. cuniculi and T. gondii among Finnish pet rabbits. We used ELISA to measure E. cuniculi IgG seroprevalence of 247 rabbits and modified direct agglutination test for T. gondii seroprevalence of 270 rabbits. Samples were collected as part of the Finnish Pet Rabbit Health Research project. Internet-based questionnaires (n = 231) completed by the rabbit owners were used for risk-factor analysis. RESULTS: The apparent seroprevalence of E. cuniculi was 29.2% and true seroprevalence of T. gondii 3.9%. Risk factors were analysed only for E. cuniculi due to the low T. gondii seroprevalence. The final multivariable logistic regression model revealed that rabbits spending the whole summer outdoors had a higher risk of being E. cuniculi seropositive than rabbits with limited outdoor access. Additionally, rabbits living in households with only one or two rabbits had higher risk of being E. cuniculi seropositive than those in multi-rabbit households. CONCLUSIONS: Nearly one third of Finnish pet rabbits participating in this study had E. cuniculi IgG antibodies, indicating previous exposure to this pathogen. The prevalence is similar to that reported previously in clinically healthy rabbit populations in UK and Korea. While the seroprevalence of T. gondii was low (3.9%), antibodies were detected. Therefore, these zoonotic parasitic infections should be considered as differential diagnoses when treating rabbits.

The first Linguatula serrata case in an imported dog in Finland.

Linguatula serrata is a pentastomid parasite infecting carnivores as definitive hosts and herbivores as intermediate hosts. In carnivores, including dogs, it usually parasitises the nasal cavity and sinuses, causing upper respiratory signs. This case report presents the first canine Linguatula case in Finland in an imported dog originating from Spain. In addition to the unremarkable clinical history of the dog, the treatment, parasite's morphology and molecular analysis are described, and the zoonotic potential is discussed.

Risk factors for equine intestinal parasite infections and reduced efficacy of pyrantel embonate against Parascaris sp.

Gastrointestinal parasites, Parascaris sp. and strongyles, are common in young horses worldwide and control of these parasites is challenged by increasing anthelmintic resistance. Our aim was to identify risk factors for these infections as well as to assess the efficacy of fenbendazole (dose 7.5 mg/kg) and pyrantel embonate (dose 19 mg/kg) against Parascaris sp. We also evaluated association between owner observed symptoms and patent infections with these parasites. Fecal samples were collected from 367 young horses in Finland and a questionnaire study was conducted. Fecal egg counts were performed by Mini-FLOTAC® method. Univariable logistic regression models using patent infection status (Yes/No), separately for Parascaris sp. and strongyle infections as an outcome were run initially to screen potential risk factors collected by the questionnaire. After the initial screening, multiple logistic regression models were constructed and run to account for correlated data structure, risk factors and potential confounders simultaneously. Two significant risk factors for a patent Parascaris sp. infection were found: breeding farm size (p = 0.028) and frequency of horse movements (p = 0.010). Horses originating from large breeding farms were more likely (OR = 2.47, 95% confidence interval (CI) 1.10-5.51) to shed Parascaris sp. eggs upon relocation to training stables compared to horses originating from small breeding farms. Horses living in farms with frequent horse movements to other premises had higher odds (OR = 3.56, 95% CI: 1.35-9.39) of a patent Parascaris sp. infection compared to farms with less frequent horse movements. Risk factors for patent strongyle infection included age (p < 0.001) and season (p = 0.017). Horses were less likely (OR = 0.27, 95% CI: 0.10 - 0.66) to shed strongylid eggs during the spring compared to the winter. Horses excreting over 200 ascarid eggs per gram were included in the anthelmintic efficacy trial. A mean FECR less than 90% was interpreted as presence of anthelmintic resistance. The mean FECR was 98.5% (95% CI: 95.8-100) and 68.0% (95% CI: 52.7-83.3) in the fenbendazole (n = 31) and pyrantel (n = 26) treatment groups, respectively. In conclusion, we identified two new risk factors for patent Parascaris sp. infection; breeding farm size and frequency of horse movements. Reduced efficacy of pyrantel against Parascaris sp. was observed for the second time in Europe. A relatively high Parascaris sp. prevalence in yearlings (34%) and two-year-olds (20%) was observed, which has not been reported earlier. An association between symptoms and a patent Parascaris sp. infection was observed in foals.

Berries as a potential transmission vehicle for taeniid eggs.

Potential role of wild forest berries as a transmission vehicle for taeniid eggs was examined using non-zoonotic Taenia laticollis eggs as a model. The berries studied were bilberries (Vaccinium myrtillus) (1 m2 plot, n = 10) and lingonberries (Vaccinium vitis-idaea) (1 m2 plot, n = 11). The plots in the managed forest were evenly sprayed with 30,000 or 60,000 T. laticollis eggs suspended in water, and berries were collected 24 h after spraying. The berries were rinsed with water, and the water was sieved through a 1-mm and a 63-µm sieve to remove coarse material and through a 20-µm sieve to collect possible eggs. A small proportion of the sieved material was examined by microscopy after treatment with fluorescent Calcofluor White stain, which binds to eggshell chitin. In the recovery tests in artificially spiked samples, the detection limit was 5 eggs in 100 g of commercial frozen bilberries and lingonberries. Taeniid eggs were detected in all of the 10 experimentally contaminated bilberry samples and in 10 of 11 lingonberry samples. The sieved debris was also analyzed for T. laticollis DNA using semi-quantitative PCR. All samples were positive in quantitative SYBR Green real-time PCR using a T. laticollis-specific primer pair amplifying a short fragment of mitochondrial NADH dehydrogenase subunit 1 gene. This indicates that forest berries contaminated in shrubs contained T. laticollis eggs, and that berries can serve as a vehicle for taeniid eggs and may pose a possible risk to humans.

A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus).

Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10-5 ng/µl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables.

Immunoproteomics and Surfaceomics of the Adult Tapeworm Hymenolepis diminuta.

In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.

Parasite infections and their risk factors in foals and young horses in Finland.

One-hundred-and-thirty-nine fecal samples were examined to assess the prevalence of Parascaris spp. and strongyle infections in two-year-old or younger horses in Finland. The owners of the horses were asked to answer an online questionnaire about the horses' environment and the management practices of the stable. The results of fecal examination and the survey were analyzed to evaluate the effect of different risk factors as ascertained by the survey on parasite prevalence. The prevalence of Parascaris spp. infections at 11.5% was lower than expected based on previous research and the strongyle prevalence of 57.6% was found in young Finnish horses. Strongyloides westeri and Eimeria leuckarti infections were also found. Pasture hygiene had a stronger influence on the prevalence of strongyle infections than on Parascaris spp. infections, whereas the hygiene routine of the horses' housing was found to be more important in the prevention of Parascaris spp. infections. The planning of the control of parasitic infections should be based on the identified risk factors.

High prevalence of zoonotic trematodes in roach (Rutilus rutilus) in the Gulf of Finland.

The intention to increase roach (Rutilus rutilus) consumption is in focus for ecological and economic reasons in Finland. However, its safety as food has not been considered comprehensively. We collected and artificially digested 85 roach halves originating from the south-eastern coast of Finland, and found trematode metacercariae in 98.8% of the samples. Based on polymerase chain reaction (PCR) and sequencing of amplicons generated from the ITS2 gene region, zoonotic parasites of the family Opistorchiidae were identified as Pseudamphistomum truncatum and Metorchis bilis, and also non-zoonotic Holostephanus dubinini (family Cyathocotylidae) and Posthodiplostomum spp. (family Diplostomidae) were identified. The species identity of other trematodes found is currently being investigated. Mixed infections of several trematode species were common. The prevalence of morphologically identified zoonotic P. truncatum was 46%, and zoonotic M. bilis was found in one sequence sample. The high prevalence of zoonotic trematode metacercariae in roach from the Gulf of Finland is alarming. Only thoroughly cooked roach products can be recommended for human or animal consumption from the area.

Identification of immunogenic proteins of the cysticercoid of Hymenolepis diminuta.

BACKGROUND: A wide range of molecules are used by tapeworm metacestodes to establish successful infection in the hostile environment of the host. Reports indicating the proteins in the cestode-host interactions are limited predominantly to taeniids, with no previous data available for non-taeniid species. A non-taeniid, Hymenolepis diminuta, represents one of the most important model species in cestode biology and exhibits an exceptional developmental plasticity in its life-cycle, which involves two phylogenetically distant hosts, arthropod and vertebrate. RESULTS: We identified H. diminuta cysticercoid proteins that were recognized by sera of H. diminuta-infected rats using two-dimensional gel electrophoresis (2DE), 2D-immunoblotting, and LC-MS/MS mass spectrometry. Proteomic analysis of 42 antigenic spots revealed 70 proteins. The largest number belonged to structural proteins and to the heat-shock protein (HSP) family. These results show a number of the antigenic proteins of the cysticercoid stage, which were present already in the insect host prior to contact with the mammal host. These are the first parasite antigens that the mammal host encounters after the infection, therefore they may represent some of the molecules important in host-parasite interactions at the early stage of infection. CONCLUSIONS: These results could help in understanding how H. diminuta and other cestodes adapt to their diverse and complex parasitic life-cycles and show universal molecules used among diverse groups of cestodes to escape the host response to infection.

Comparison of commercial ELISA tests for the detection of Toxoplasma antibodies in the meat juice of naturally infected pigs.

Toxoplasmosis is a globally distributed protozoal zoonosis. Pigs are considered an important reservoir of Toxoplasma gondii and pork a major infection source of human toxoplasmosis. ELISA methods are commonly used diagnostic tools for detecting Toxoplasma infections. They are also used for slaughterhouse-based serological monitoring of toxoplasmosis in pigs to identify positive farms. The methods used are non-standardised with varying sensitivity and specificity. In our study, four commercial ELISA tests for the detection of Toxoplasma antibodies in the meat juice of slaughter pigs were compared with a modified agglutination test (MAT) as a reference. The cut-off values of the ELISA tests provided by the manufacturer varied between 0.20 and 0.50, and clearly influenced prevalence. The sensitivity of tests I, II and III varied between 96.4 and 78.6. Sensitivity was unacceptably low (3.6) for test IV (cut-off=0.30). Tests I, II and III had the highest accuracy and the best agreement with the reference test when a cut-off of 0.30 was used. Test II and III showed very good agreement (K=0.92 and 0.84, respectively) with the MAT. A very strong correlation (Pearson correlation >0.89) was observed between the S/P values of tests I, II and III. Our results demonstrate that the test and cut-off value used influence the results of the apparent seroprevalence studies.

Comparative Proteomic Analysis of Hymenolepis diminuta Cysticercoid and Adult Stages.

Cestodiases are common parasitic diseases of animals and humans. As cestodes have complex lifecycles, hexacanth larvae, metacestodes (including cysticercoids), and adults produce proteins allowing them to establish invasion and to survive in the hostile environment of the host. Hymenolepis diminuta is the most commonly used model cestode in experimental parasitology. The aims of the present study were to perform a comparative proteomic analysis of two consecutive developmental stages of H. diminuta (cysticercoid and adult) and to distinguish proteins which might be characteristic for each of the stages from those shared by both stages. Somatic proteins of H. diminuta were isolated from 6-week-old cysticercoids and adult tapeworms. Cysticercoids were obtained from experimentally infected beetles, Tenebrio molitor, whereas adult worms were collected from experimentally infected rats. Proteins were separated by GeLC-MS/MS (one dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry). Additionally protein samples were digested in-liquid and identified by LC-MS/MS. The identified proteins were classified according to molecular function, cellular components and biological processes. Our study showed a number of differences and similarities in the protein profiles of cysticercoids and adults; 233 cysticercoid and 182 adult proteins were identified. From these proteins, 131 were present only in the cysticercoid and 80 only in the adult stage samples. Both developmental stages shared 102 proteins; among which six represented immunomodulators and one is a potential drug target. In-liquid digestion and LC-MS/MS complemented and confirmed some of the GeLC-MS/MS identifications. Possible roles and functions of proteins identified with both proteomic approaches are discussed.

Prevalence of intestinal parasites and risk factor analysis for Eimeria infections in Finnish pet rabbits.

No previous published prevalence studies exist on gastrointestinal parasites in Finnish pet rabbits; internationally, similar prevalence figures remain uncommon. This study aimed to estimate the prevalence of gastrointestinal parasites in pet rabbits as well as to determine the possible risk factors for parasitic infections. We analyzed 2-g faecal samples (n=398) from pet rabbits and internet-based questionnaires (n=363) completed by their owners. Owners sent over-night faecal samples to the laboratory, and the samples were quantitatively analyzed within one week using a modified McMaster method. Eimeria oocysts represented the most common parasite found (27%, mean opg 4212). Nematode Passalurus ambiguus eggs were found in 3% of the samples (mean epg 65), while Trichuris leporis eggs and cestode eggs, respectively, were each found in 1 sample (0.25%). We also conducted a risk factor analysis based on the owner questionnaire and the faecal analysis. We limited this to only Eimeria infection due to the low number of positive results for other parasites. In the final multivariable logistic regression model, we identified a young age, multi-rabbit households (with at least three rabbits) and living somewhere other than in a home-like environment as risk factors for Eimeria infection. In similar low-helminth prevalence conditions, we recommend faecal examination and deworming of rabbits according to examination results.

Mass spectrometry analysis of the excretory-secretory (E-S) products of the model cestode Hymenolepis diminut a reveals their immunogenic properties and the presence of new E-S proteins in cestodes.

Hymenolepis diminuta is an important model species in studies of therapeutics, biochemical processes, immune responses and other aspects of cestodiasis. The parasite produces numerous excretory-secretory (E-S) proteins and a glycocalyx covering its body. Our study focused on the mass spectrometry analysis of the E-S material with an objective to determine if E-S contains any new proteins, in particular those that can be identified as: antigens, vaccine candidates and drug targets. These proteins might engage directly in host-parasite interactions. Adult parasites collected from experimentally infected rats were cultured in vitro for 5 and 18h. Immunoblotting was used to verify which E-S protein bands separated in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) react with specific antibodies from sera of infected rats. We identified thirty-nine proteins by LC-MS/MS (liquid chromatography mass spectrometry). Results indicated the presence of proteins that have never been identified in cestode E-S material. Immunoblotting showed the immunogenicity of E-S products of H. diminuta, most probably associated with the presence of proteins known as antigens in other flatworm species. Among identified proteins are those engaged in immunomodulatory processes (eg. HSP), in response to oxidative stress (peroxidasin) or metabolism (eg. GAPDH). The predominant functions are associated with metabolism and catalytic activity. This is the first study identifying E-S-proteins in adult tapeworms, thus providing information for better understanding host-parasite interrelationships, and may point out potential targets for vaccines or drug discovery studies, as among the proteins observed in our study are those known to be antigens.

Reappraisal of Hydatigera taeniaeformis (Batsch, 1786) (Cestoda: Taeniidae) sensu lato with description of Hydatigera kamiyai n. sp.

The common cat tapeworm Hydatigera taeniaeformis is a complex of three morphologically cryptic entities, which can be differentiated genetically. To clarify the biogeography and the host spectrum of the cryptic lineages, 150 specimens of H. taeniaeformis in various definitive and intermediate hosts from Eurasia, Africa and Australia were identified with DNA barcoding using partial mitochondrial cytochrome c oxidase subunit 1 gene sequences and compared with previously published data. Additional phylogenetic analyses of selected isolates were performed using nuclear DNA and mitochondrial genome sequences. Based on molecular data and morphological analysis, Hydatigera kamiyai n. sp. Iwaki is proposed for a cryptic lineage, which is predominantly northern Eurasian and uses mainly arvicoline rodents (voles) and mice of the genus Apodemus as intermediate hosts. Hydatigera taeniaeformis sensu stricto (s.s.) is restricted to murine rodents (rats and mice) as intermediate hosts. It probably originates from Asia but has spread worldwide. Despite remarkable genetic divergence between H. taeniaeformis s.s. and H. kamiyai, interspecific morphological differences are evident only in dimensions of rostellar hooks. The third cryptic lineage is closely related to H. kamiyai, but its taxonomic status remains unresolved due to limited morphological, molecular, biogeographical and ecological data. This Hydatigera sp. is confined to the Mediterranean and its intermediate hosts are unknown. Further studies are needed to classify Hydatigera sp. either as a distinct species or a variant of H. kamiyai. According to previously published limited data, all three entities occur in the Americas, probably due to human-mediated introductions.

Innovative molecular diagnosis of Trichinella species based on ß-carbonic anhydrase genomic sequence.

Trichinellosis is a helminthic infection where different species of Trichinella nematodes are the causative agents. Several molecular assays have been designed to aid diagnostics of trichinellosis. These assays are mostly complex and expensive. The genomes of Trichinella species contain certain parasite-specific genes, which can be detected by polymerase chain reaction (PCR) methods. We selected ß-carbonic anhydrase (ß-CA) gene as a target, because it is present in many parasites genomes but absent in vertebrates. We developed a novel ß-CA gene-based method for detection of Trichinella larvae in biological samples. We first identified a ß-CA protein sequence from Trichinella spiralis by bioinformatic tools using ß-CAs from Caenorhabditis elegans and Drosophila melanogaster. Thereafter, 16 sets of designed primers were tested to detect ß-CA genomic sequences from three species of Trichinella, including T. spiralis, Trichinella pseudospiralis and Trichinella nativa. Among all 16 sets of designed primers, the primer set No. 2 efficiently amplified ß-CA genomic sequences from T. spiralis, T. pseudospiralis and T. nativa without any false-positive amplicons from other parasite samples including Toxoplasma gondii, Toxocara cati and Parascaris equorum. This robust and straightforward method could be useful for meat inspection in slaughterhouses, quality control by food authorities and medical laboratories.

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries.

BACKGROUND: Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries. RESULTS: In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out. Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs. CONCLUSIONS: This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.

Early Trichinella spiralis and Trichinella nativa infections induce similar gene expression profiles in rat jejunal mucosa.

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ≤ 0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella.