Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

The free radical nitric oxide (NO) and Ca2+ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

Dirofilaria Immitis in Bulgaria: The First Genetic Baseline Data and an Overview of the Current Status.

Dirofilaria immitis, the agent of canine dirofilariosis, is a common parasite of domestic and wild carnivores with zoonotic potential and worldwide distribution, being endemic in many countries. Bulgaria is among European countries recognized as endemic for this heartworm parasite. In the present study, D. immitis adults recovered from pulmonary arteries of domestic dog and golden jackal originating from the Pazardzhik region in southern Bulgaria, and from red fox originating from the Plovdiv region in central-southern Bulgaria, were genetically analyzed in nuclear targets. The first PCR amplification of the internal transcribed region 2 (ITS2) of the ribosomal DNA with previously published D. immitis-specific primers yielded single fragments in size of 302 bp that is characteristic for these heartworms. PCR products of three isolates, resulted from the second amplification of the 5.8S-ITS2 region (235 bp) with pan-filarioid primers, were subjected to direct DNA sequencing. Identical nucleotide composition was detected across the screened target region for these Bulgarian isolates. When the 5.8S-ITS2 sequences were phylogenetically compared to the GenBank-retrieved D. immitis sequences in a worldwide context, the neighbor-joining analysis has shown three discrete clades. The first clade was composed of D. immitis isolates from Europe (including the studied Bulgarian samples), Asia and South America, in the second clade samples from Asia and South America were placed, whereas the third clade was formed by two Brazilian dog isolates originated from the north and southeast part of the country. The purpose of the present study was to verify the taxonomic characterization of D. immitis nematodes from Bulgaria based on morphology and compare their genetic structure with filariae obtained from the different world regions using molecular assays. It also summarizes previous epidemiological and ecological studies on the parasite distribution and prevalences in different hosts and regions undertaken so far in Bulgaria.

Probiotic Bacteria can Modulate Murine Macrophage's Superoxide Production in Trichinella Spiralis Infection.

The effect of probiotic strains (Enterococcus faecium EF55, E. faecium CCM7420, E. faecium CCM8558, E. durans ED26E/7, Lactobacillus fermentum CCM7421, L. plantarum 17L/1) on the production of superoxide anion (O2 -) in peritoneal macrophages of Trichinella spiralis infected mice was examined. E. faecium EF55 and E. faecium CCM8558 strains increased the O2 -production prior to parasitic infection,at the day7of application.A significant inhibition of the O2 - production caused by T. spiralis infection on day 5 post infection (p.i.) was prevented by all examined strains. Lactobacilli stimulated metabolic activity of macrophages during intestinal and early muscular phase (from day 5 to 25 p.i.) of trichinellosis. Enterococci increased the O2 - production in early intestinal phase (day 5 p.i.) and during the muscular phase of trichinellosis (days 25 and 32 p.i.). Respected increase in macrophage's metabolic activity induced by probiotic treatment in the intestinal phase of trichinellosis augmented the host antiparasite defence (damage and killing of newborn larvae with reactive oxygen species from macrophages).

The Mouse Bile Duct Tapeworm, Hymenolepis Microstoma in Free-living Small Mammals in Slovakia: Occurrence and Genetic Analysis.

The mouse bile duct tapeworm Hymenolepis microstoma, is a potentially zoonotic species with a wide variety of reported definitive hosts of rodent genera. In the present study the occurrence of H. microstoma in free-living small mammals in selected areas of Slovakia and the retrospective analysis of epidemiological data published in Slovakia were performed. Hymenolepis microstoma was detected in two animal species, the common shrew (Sorex araneus) and the European hamster (Cricetus cricetus) of 186 small mammals examined from two ecosystems, urban and natural ecosystem of national park. No mention about the presence of this parasite in Slovakia in the past was found following a bibliographical search. Partial sequences of the nuclear paramyosin gene showed the shrew isolate placed in a subclade together with H. microstoma from Portugal, with high bootstrap value for its differentiation from the sister species Hymenolepis nana. Similarly, the analysis of the nuclear ribosomal ITS region placed the hamster isolate in the cluster composed of H. microstoma from Australia, Spain and Portugal. The Slovak isolate was the most distinctive sample among available H. microstoma, differing in 1.4 - 1.9% of nucleotides from the remaining isolates. The difference (seven of 17 nucleotide positions) was partially due to indel polymorphisms associated with two and five nucleotides. To our knowledge, these are the first reports of H. microstoma in Central Europe and also the first record of infection in the common shrew. A recently indicated zoonotic potential of H. microstoma along with a possibility of its direct transmission between animals and/or humans without the need of intermediate hosts pose a public health concern in contaminated areas of Slovakia. The use of molecular techniques may substantially facilitate more thorough understanding of the epidemiological situation of H. microstoma and related tapeworms in various ecosystems of the country.

Rare Case of Angiostrongylus Vasorum Intraocular Infestation in an Asymptomatic Dog.

The presented clinical observation shows an atypical case of Angiostrongylus vasorum intraocular infection in an 18-month-old male beagle from north-eastern Slovakia. The dog presented with a motile worm in the anterior chamber of the right eye. No ocular signs or symptoms of a systemic disease were observed. The faecal examination using Baermann´s technique and fl otation was negative. Diagnosis was established following surgical removal of the worm. The specimen was determined as an A. vasorum female based on morphological features and confirmed by means of PCR technique and sequencing. To the best of our knowledge, the presented manifestation is the first ocular case of angiostrongylosis with absence of typical symptoms or signs of the disease.

The Anti-parasitic Effect of Probiotic Bacteria via Limiting the Fecundity of Trichinella Spiralis Female Adults.

A potential protective effect of probiotic strains against zoonotic Trichinella spiralis infection was investigated in the framework of a new therapeutic strategy aimed at using probiotics to control parasitic zoonoses. The study was focused on the impact of six selected probiotic (bacteriocinogenic) strains on the intensity of T. spiralis infection and female fecundity ex vivo and in vitro. Bacterial strains of different origin (Enterococcus faecium EF55, Enterococcus faecium 2019 = CCM7420, Enterococcus faecium AL41 = CCM8558, Enterococcus durans ED26E/7, Lactobacillus fermentum AD1 = CCM7421, Lactobacillus plantarum 17L/1) were administered daily in a dose of 109 CFU/ml in 100 µl, and mice were infected with 400 T. spiralis larvae on day 7 of treatment. Female adults of T. spiralis were isolated on day 5 post infection (p.i.) and subsequently were used in fecundity test ex vivo. E. faecium CCM8558, E. faecium CCM7420 and E. durans ED26E/7 strains significantly reduced the number of adults in the intestine. The application of L. fermentum CCM7421, L. plantarum 17L/1, E. faecium CCM8558 and E. durans ED26E/7 caused a significant decrease in the number of muscle larvae. The treatment with E. faecium CCM8558 and E. durans ED26E/7 showed the highest inhibitory effect on female fecundity (94 %). The number of newborn larvae (NBL) was also significantly decreased after administration of L. fermentum CCM7421 and L. plantarum 17L/1 (80 %). A direct impact of probiotic strains on female reproductive capacity was examined in vitro in females isolated from untreated infected mice on day 5 p.i. A correlation was found between the inhibitory effect and the concentration of probiotic strains. The reduction effects of the strains manifested as follows: L. fermentum CCM7421 (93 %), E. faecium CCM8558, L. plantarum 17L/1, E. faecium EF55 (about 80 %), E. faecium CCM7420 and E. durans ED26E/7 (about 60 %).

Seroprevalence of Trichinella infections in domestic dogs from Slovakia.

Trichinellosis is constantly present in the sylvatic cycle in Slovakia, with several sporadic human outbreaks registered since the 1960s. The largest outbreak of trichinellosis occurred in 1998 and was related to the consumption of dog meat that had been added to smoked pork sausages. The aim of this study was to estimate the seroprevalence of Trichinella infection in domestic dogs in various regions of Slovakia. Out of 439 dogs tested, 56 (12.8%) were classified as a seropositive based on the enzyme-linked immunosorbent assay (ELISA), with confirmation by Western blotting. The highest seropositivity was recorded among dogs from the eastern part of Slovakia, in the Presov (22.9%) and Kosice (17.1%) regions, long considered to be highly endemic for Trichinella occurrence and where the prevalence of infection in the vulpine population is also the highest in Slovakia. Trichinella-seropositive animals were detected significantly more often in dogs kept in rural areas (21.1%) when compared with animals living in towns or suburban localities (7.7%). On the other hand, neither age, nor gender and size of the dogs were shown to be a significant factor for Trichinella infection (P > 0.05). The present results indicate that the dog population in Slovakia is at high risk of exposure to Trichinella parasites and could potentially represent a suitable host group for serological monitoring of Trichinella infection as convenient sentinel animals.

Acanthocephalans of the genus Centrorhynchus (Palaeacanthocephala: Centrorhynchidae) of birds of prey (Falconiformes) and owls (Strigiformes) in Slovakia.

Three species of thorny-headed worms of the genus Centrorhynchus were found to parasitize birds of prey and owls in the territory of the Slovakia during the years 2012-2014. Out of 286 examined bird individuals belonging to 23 species, only Buteo buteo, Buteo rufinus, Falco tinnunculus (Falconiformes), Asio otus, Strix aluco, Strix uralensis and Tyto alba (Strigiformes) were infected by acanthocephalans. All the bird species except for S. aluco represent new host records for Slovakia. The most prevalent acanthocephalan Centrorhynchus aluconis was detected in all 15 examined birds of non-migratory Ural owl S. uralensis (P = 100%); however, it was found occasionally also in two individuals of the tawny owl S. aluco (P = 20%), one long-eared owl A. otus (P = 7.7%), one barn owl T. alba (P = 33.3%) and the common buzzard B. buteo (P = 0.8%). Two other thorny-headed worms occurred exclusively in Falconiformes in raw or mixed infections: Centrorhynchus buteonis was found in 11 individuals of B. buteo (P = 9.2%), and two birds (B. buteo and B. rufinus) were parasitized simultaneously by C. buteonis and the species Centrorhynchus globocaudatus. Moreover, the latest, relatively rare acanthocephalan was found alone in two common kestrels F. tinnunculus (P = 2.7%). Regarding intensity of infection, it ranged from a single female of C. buteonis, C. globocaudatus or C. aluconis per host (four cases) to a maximum of 82 C. aluconis per an Ural owl. The difference in acanthocephalan species spectrum between birds of prey and owls in Slovakia was apparent.

First autochthonous case of canine Angiostrongylus vasorum in Slovakia.

Angiostrongylus vasorum is a metastrongyloid nematode that may cause cardiopulmonary disease, neurological signs and coagulopathies in dogs. The parasite has an indirect life cycle with molluscs as intermediate hosts, in which the infective third larval stage develops. Recently, A. vasorum has been repeatedly reported in dogs outside the endemic areas, indicating that this parasite is widely distributed over Europe. This is the first record of an autochthonous infection in a dog from Slovakia that was casually diagnosed during routine preventive parasitological examination. A. vasorum first-stage larvae were recovered using the Baermann technique and identified by length and characteristic tail morphology. The animal originated from Slovakia and had not travelled abroad. The dog had been regularly walked on grass fields with a concentrated presence of common species of Gastropoda and frogs. The owner reported that the dog had been licking and eating grass and it had shown curiosity for molluscs and frogs. The first finding of A. vasorum-infected dog in Slovakia has confirmed that the parasite is spreading beyond the traditional hyperendemic foci, which accentuates the need for monitoring and increasing of disease awareness in primary care clinical practice.

Molecular evidence for the presence of Dirofilaria repens in beech marten (Martes foina) from Slovakia.

Herein we present the first finding of Dirofilaria repens, agent of the subcutaneous form of dirofilariosis, in Martes foina. Molecular analyses from the spleen of 3 individuals originated from Tatra National Park, Northern Slovakia, confirmed the presence of D. repens in one of them. Finding of D. repens in beech marten instigates to more intense research on free living carnivores as the potential source of Dirofilaria parasites.

Dirofilaria infections in working dogs in Slovakia.

A monitoring programme aimed at the diagnosis of subcutaneous dirofilariasis and heartworm disease in working (police and military) dogs in Slovakia has been performed during the period of September 2007 to February 2008. In co-operation with the Ministry of the Interior and the Ministry of Defence, in total, 710 dogs (591 police dogs and 119 military dogs) were investigated for the presence of microfilariae in blood. All police and military dogs in active service held on the territory of Slovakia were included. Microfilariae were detected in 118 (20.0%) police dogs and 10 (8.4%) military dogs. The most infected individuals originated from southern parts of Slovakia (Trnava region 53.6% and Nitra region 39.6%); the prevalence was low in northern regions (Zilina 3.1% and Presov 6.6%). In several districts of southern Slovakia, the prevalence of subcutaneous dirofilariasis in working dogs exceeded 40%. In all infected animals, the autochthonous origin of the disease was confirmed; however, due to the frequent movement of working dogs, it was not possible to identify the exact locality of infection. At present, a dog living in Nemsová village in Trencín district (north-western part of the country) is regarded as the northernmost localized autochthonous case of subcutaneous dirofilariasis in Slovakia. In three dogs, co-infection of Dirofilaria repens and Dirofilaria immitis was detected. High prevalence rates in working dogs and the zoonotic characteristic of the disease represent an undoubtedly important veterinary and medical problem that requires the urgent introduction of prophylactic and control measures.

Long-term survey on Trichinella prevalence in wildlife of Slovakia.

In Slovakia, monitoring the prevalence of Trichinella spp. in wildlife was performed since 2000 in the main reservoir animals, the red fox (Vulpes vulpes) and wild boar (Sus scrofa), using artificial digestion method as recommended by International Commission on Trichinellosis. The results of investigation performed in 5270 red foxes showed that Trichinella infection is widespread across Slovakia and prevalence increased significantly from 4.9% in 2000 to 20.5% in 2007. Recently, a higher Trichinella prevalence (0.11%) in wild boars was also demonstrated. The results indicate that foxes and wild boars are involved in the spread of Trichinella, although the latter host species seems to play a secondary role in the maintenance of the sylvatic cycle in Slovakia. Trichinella britovi is the predominant species circulating in Slovakia, both in foxes and wild boars, and Trichinella spiralis occurs only sporadically. Mixed infections of T. britovi and Trichinella pseudospiralis were recorded in 2005 in one wild boar from Eastern Slovakia and in 2006 in one red fox from the same region. These findings are important with respect to an outbreak caused by T. pseudospiralis in a pig farm in the same district 3 years ago. This study provides a complex picture on Trichinella occurrence in all regions of Slovakia and may be a good basis for evaluating the risk of parasite transmission to the domestic cycle and human beings.

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae.

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20 dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30 dpi. The IgG antibodies appeared at 40 dpi in inbred mice, and at 50 dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30 dpi. Antibody levels increased until the end of the experiment (80 dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.

Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions.

The study of the genetic polymorphism of pathogens is important for phylogenetic and biogeographic studies and, in the case of foodborne pathogens, to trace the origin of food infection. Since its discovery in 1972, the nonencapsulated species Trichinella pseudospiralis has been detected in mammals and birds, and human infection has occurred, in some cases resulting in death. We studied DNA polymorphism among ten T. pseudospiralis isolates from the Palearctic, Nearctic, and Australian regions, screening the sequences of nine genes [18sRNA, a random amplified polymorphism DNA derived sequence, mitochondrial cytochrome oxidase subunit I (COI), cytochrome P450, cynate lyase, epithelial fusion failure-1, and three unknown genes of Tp3, Tp8, and Tp26]. A high identity of sequence for the nine gene loci was obtained among the seven isolates from the Palearctic region and between the two isolates from the Nearctic region. Genetic identity analysis indicated the distinct polymorphism among the three geographical origins. To easily identify T. pseudospiralis genotypes, a polymerase chain reaction-restriction fragment length polymorphism analysis of COI gene was performed, and the results confirmed the DNA polymorphism within T. pseudospiralis, corresponding to the three regions of origin. We have named the three genotypes as "T. pseudospiralis Palearctic genotype" (code T4P), "Nearctic genotype" (code T4N), and "Australian genotype" (code T4A). To further investigate polymorphism among the nonencapsulated Trichinella species, the sequences of four gene loci (COI, P450, cynate lyase, and SB147D) of T. pseudospiralis, T. papuae, and T. zimbabwensis were analyzed, and the results showed high polymorphism among the three species, strongly supporting their classification as separate species.

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.