Determining the pathogenicity and virulence of parasites on animal models.

The aim of this review is to present various animal organisms used to determine the pathogenicity and virulence of old and new human and animal pathogens based on animal studies, cell cultures, macrophages and other models. The animal models presented in this study, in addition to the most popular organisms such as the laboratory mouse, rat, guinea pig and monkey, are also less popular models, such as zebrafish (Danio rerio) or chicken embryos in eggs. These animals are used to study the pathogenicity of parasites such as Acanthamoeba, Naegleria fowlerii, Toxoplasma gondii, Entamoeba histolytica and Besnoitia caprae and other species. In addition to animal models, we also present models using cell cultures, macrophages and computer methods. We also answer questions about what experimental methods allow to differentiate species and populations in terms of pathogenicity and virulence.

The use of phytotherapy in the fight against parasitic diseases.

In recent years, there has been more and more new research on the therapeutic effects of plants and their positive impact on the fight against parasitic diseases. It is of great importance, as it gives the opportunity to use this knowledge for phytotherapy, which is cheaper than pharmacological treatment, and as numerous studies have shown, it can be equally effective. Scientists are still looking for newer and newer chemicals that can be isolated from plants around us, and the current medicine is more and more willing to use natural medicines. In the following work, we present an overview of the most common parasitic diseases caused by protozoa, flatworms, roundworms, as well as by arachnids and fleas. We also presented alternative methods of treating these diseases using phytotherapy, which uses extracts of, among others, mint, tea tree, garlic, ginger, pumpkin seeds, annual mugwort, musk cosmos, walnuts, cocoa, grapes or black cumin.

Changes in the Expression of TLR2 During the Intestinal Phase of Trichinellosis.

INTRODUCTION: Toll-like receptors (TLRs) play an important role in fast activation of the immune response to a variety of pathogens, including parasites. In this study, we focused on TLR2, because this receptor is one of the best known and most frequently analysed members of the TLR family. The aim of this study was to assess the effect of Trichinella spiralis on expression of TLR2 during the intestinal stage of infection. MATERIAL AND METHODS: The experimental material consisted of isolates prepared from the intestines (jejunum and colon) of BALB/c mice infected with T. spiralis taken at 4, 8, and 16 days post infection. RESULTS: Our results based on quantitative real-time PCR showed that the mRNA level for TLR2 was statistically significantly higher in the jejuna of mice infected with T. spiralis than in this tissue of uninfected mice. In addition, the presence of TLR2 protein in the intestinal phase of trichinellosis was confirmed by a strong positive immunohistochemical reaction. CONCLUSION: Our results indicate that infection with T. spiralis changes the expression of TLR2 in the small intestine of the mouse host and suggest a contribution of these receptors to the host defence mechanisms during experimental trichinellosis.

Phytochemical Screening and Acanthamoebic Activity of Shoots from in Vitro Cultures and in Vivo Plants of Eryngium alpinum L.-The Endangered and Protected Species.

Genetically uniform shoots of Eryngium alpinum L. cultured in vitro were subjected to the qualitative analysis applying the UPLC-HESI-HRMS technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. The phytochemical screening of the crude methanolic extracts of shoots, both from in vitro cultures and in vivo plants, revealed the presence of phenolic acids, coumarins, flavonoids, triterpenoid saponins, amino acids, or dipeptides. Active compounds detected are known to have medicinal importance, and for this reason, the present study represents a preliminary investigation of the extracts against pathogenic and opportunistic amoeba. Among the extracts tested, the extract of shoots from in vitro cultures exhibited remarkable amoebicidal action against trophozoites. On the second day of treatment, the extract at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL showed the highest antiamoebicidal effect: the inhibition of trophozoites reached 81.14%, 66.38%, and 54.99%, respectively. To our best knowledge, the present report is the first to show the phytochemical screening and to discuss the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.

The modulatory effect of Artemisia annua L. on toll-like receptor expression in Acanthamoeba infected mouse lungs.

The genus Acanthamoeba, which may cause different infections in humans, occurs widely in the environment. Lung inflammation caused by these parasites induces pulmonary pathological changes such as pulmonary necrosis, peribronchial plasma cell infiltration, moderate desquamation of alveolar cells and partial destruction of bronchial epithelial cells, and presence of numerous trophozoites and cysts among inflammatory cells. The aim of this study was to assess the influence of plant extracts from Artemisia annua L. on expression of the toll-like receptors TLR2 and TLR4 in lungs of mice with acanthamoebiasis. A. annua, which belongs to the family Asteraceae, is an annual plant that grows wild in Asia. In this study, statistically significant changes of expression of TLR2 and TLR4 were demonstrated. In the lungs of infected mice after application of extract from A. annua the expression of TLRs was observed mainly in bronchial epithelial cells, pneumocytes (to a lesser extent during the outbreak of infection), and in the course of high general TLR expression. TLR4 in particular was also visible in stromal cells of lung parenchyma. In conclusion, we confirmed that a plant extract of A. annua has a modulatory effect on components of the immune system such as TLR2 and TLR4.

Influence of Artemisia annua L. on toll-like receptor expression in brain of mice infected with Acanthamoeba sp.

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool.

Trichinella Spiralis: Impact on the Expression of Toll-like Receptor 4 (TLR4) Gene During the Intestinal Phase of Experimental Trichinellosis.

INTRODUCTION: Toll-like receptors (TLRs) play a key role in the rapid activation of the innate immune response to a variety of pathogens. The aim of this study was to evaluate the effect of Trichinella spiralis infection on the level of expression of the tlr4 gene in mouse intestines during the intestinal phase of experimental trichinellosis. MATERIAL AND METHODS: The experimental material consisted of the small and large intestines of BALB/c mice infected with Trichinella spiralis sampled at 4, 8, and 16 days post infection (dpi). RESULTS: A statistically significant increase was demonstrated in the tlr4 mRNA level isolated from the infected mice jejunum at 4, 8, and 16 dpi over the uninfected control. Moreover, at 4, 8, and 16 dpi in the jejunum of infected mice, a strong positive reaction for the presence of TLR4 protein compared with that of uninfected mice was observed. CONCLUSION: Infection with T. spiralis changes the expression of the tlr4 gene in the small intestine of the mouse host.

Hygiene pests as vectors for parasitic and bacterial diseases in humans

Diseases transmitted by hygiene pests remain a very serious problem in spite of fast developments in science and medicine. The present study focuses on pests carrying germs that pose a threat to human health and life. The quick pace of life, the need to satisfy human needs and mass production of food sometimes result in flagrant sanitary, hygienic and epidemiological deficiencies. These irregularities are conducive to hygiene pests, which, when not held in check by proper control measures, may act more efficiently and quickly.

Evaluation of the effectiveness of tea tree oil in treatment of Acanthamoeba infection.

Eye diseases caused by amoebae from the genus Acanthamoeba are usually chronic and severe, and their treatment is prolonged and not very effective. The difficulties associated with therapy have led to attempts at finding alternative treatment methods. Particularly popular is searching for cures among drugs made of plants. However, no substances with total efficacy in treating Acanthamoeba keratitis have been identified.Results of our semi in vivo studies of tea tree oil simulating eyeball infection demonstrated 100% effectiveness in the case of both trophozoites and cysts of amoebae from the genus Acanthamoeba. The action of tea tree oil indicates that this is the first substance with a potential ability to quickly and effectively remove the amoebae from the eye. Tea tree oil has the ability to penetrate tissues, which allows it to destroy amoebae in both the shallow and deep layers of the cornea. The present research into the use of tea tree oil in the therapy of Acanthamoeba infection is the first study of this type in parasitology. It offers tremendous potential for effective treatment of Acanthamoeba keratitis and other diseases caused by these protozoa.

THE USE OF EXTRACTS FROM PASSIFLORA SPP. IN HELPING THE TREATMENT OF ACANTHAMOEBIASIS.

Chronic progressive diseases of the central nervous system such as granulomatous amoebic encephalitis, amoebic keratitis, amoebic pneumonitis and also skin infections caused by free-living amoebae (Acanhamoeba spp.) are a significant challenge for pharmacotherapy. This is due to the lack of effective treatment because of encystation, which makes the amoebae highly resistant to anti-amoebic drugs. A very inter- esting and promising source of future drugs in this area are plant materials obtained not only from the habitat but also from plant in vitro culture as an alternative source of biomaterials. Alcoholic extracts from leaves of Passiflora incarnata, P. caerulea, P. alata (Passifloraceae) and from callus cultures were evaluated in vito for amoebicidal activity. Phytochemical analysis showed that all extracts contained phenolic compounds including flavonoids? Biological study revealed that all extracts showed amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. Extracts of P. alata leaf and callus showed the most effective activities (IC5, 4.01 mg/mL, IC,5 7.29 mg/mL, respectively) after 48 h of exposure, which was correlated with the highest concentration of total phenolics and flavonoids in comparison with other extracts.

Toll-like receptors in the brain of mice following infection with Acanthamoeba spp.

The Toll-like receptors (TLRs) of the innate immune system play an important role in the recognition of pathogens such as bacteria, viruses, fungi, and parasites. In this study, we examined the changes in the level of expression of TLR2 and TLR4 mRNA and protein in the brains of mice infected with Acanthamoeba spp. The Acanthamoeba strains were isolated from a patient with Acanthamoeba keratitis (AK) (Ac55) and Malta Lake (Ac43). In the brain isolated from mice at 2 days post-infection (dpi) with Acanthamoeba strains Ac55 and Ac43, mRNAs for TLR2 and TLR4 were significantly more strongly expressed in comparison with the uninfected mice. In Acanthamoeba-infected mice, TLR2 and TLR4 expression was detected in neurons, glial cells, and endothelial cells within the neocortex. These receptors showed more intense expression in ependymocytes of the choroid plexus of infected mice at 2 dpi. Increased levels of TLR2 and TLR4 mRNA expression in infected mice suggest the involvement of these TLRs in the recognition of Acanthamoeba spp. pathogen-associated molecular patterns (PAMPs).

Acanthamoeba infection in lungs of mice expressed by toll-like receptors (TLR2 and TLR4).

Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznan, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP).

Artemisia annua L. as a plant with potential use in the treatment of acanthamoebiasis.

The treatment of acanthamoebiasis is a great problem. Most cerebral invasions end with death, and the treatment of ocular invasions is usually long-lasting and not very effective. Numerous plant extracts and substances isolated from plants, which are effective against trophozoites or cysts, have been studied in the treatment of acanthamoebiasis. However, no agents that are simultaneously effective against both developing forms of amoebae have been discovered yet. It seems that such a plant which fulfils both tasks is Artemisia annua L. Our studies showed that water, alcohol and chloroform extracts from the herb A. annua L. can be applied in general and local treatment or in combined therapy with antibiotics in the treatment of acanthamoebiasis. Extracts from this plant show not only in vitro but also in vivo effects. Studies carried out on experimental animals infected with amoebae show that the application of these extracts significantly prolongs the survival of the animals.

Parasitic diseases in humans transmitted by vectors.

Despite the considerable progress of medicine, parasitic diseases still pose a great threat to human health and life. Among parasitic diseases, those transmitted by vectors, mainly arthropods, play a particular role. These diseases occur most frequently in the poorest countries and affect a vast part of the human population. They include malaria, babesiosis, trypanosomiasis, leishmaniasis and filariasis. This study presents those vector-transmitted diseases that are responsible for the greatest incidence and mortality of people on a global scale. Attention is focused primarily on diseases transmitted by mosquitoes, flies, Hemiptera and ticks.

Genotypic characterization of amoeba isolated from Acanthamoeba keratitis in Poland.

Free-living amoebae belonging to the genus Acanthamoeba are the causative factor of many diseases. Among others, they cause Acanthamoeba keratitis (AK), a condition that usually occurs in contact lens wearers, though it is also observed in non-wearers. The number of diagnosed cases of AK increased more than eightfold during 8 years in the USA, and a proportional increase in frequency also occurred in Poland and Europe. Cases of AK are usually diagnosed late, and their therapy is difficult and rarely successful. AK is an uncommon diagnosis in Poland. The increased number of positive cases observed in our laboratory may reflect the growing at-risk population of contact lens wearers. Acanthamoeba as a genus of facultative human parasites is currently classified into 17 genotypes. Isolates belonging to seven genotypes were found to be associated with AK. One genotype in particular, T4, was found to be overrepresented in human disease. The main finding of our study is that in Poland, AK is almost always associated with the T4 genotype.

Abietane diterpenoids from Salvia sclarea transformed roots as growth inhibitors of pathogenic Acanthamoeba spp.

Amoebae from the genus Acanthamoeba are known agents leading to various diseases such as granulomatous amoebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumitis (AP), chronic lung infection, and skin infections. It is known that various synthetic anti-Acanthamoeba substances are ineffective. Therefore, other substances, e.g., natural plant compounds, are the focus of biological investigations regarding anti-parasite activity. In this work, the ability of four abietane diterpenoids (ferruginol, salvipisone, aethiopinone, and 1-oxo-aethiopinone) to inhibit Acanthamoeba growth is reported. All investigated compounds were active against Acanthamoeba growing in vitro. Among them, ferruginol demonstrated the highest activity against Acanthamoeba. This compound inhibited Acanthamoeba growth by about 72% in a 3-day exposure period (IC50 17.45 µM), while aethiopinone and 1-oxo-aethiopinone demonstrated this activity at the level of 55-56%. Salvipisone reduced the growth of Acanthamoeba in vitro culture by 39%. For this compound, the value of IC50 was 701.94 µM after 72 h of exposure.

Comparative analyses of different genetic markers for the detection of Acanthamoeba spp. isolates.

Acanthamoeba are widespread free-living amoebae which may cause granulomatous amoebic encephalitis (GAE), keratitis, skin ulcerations and disseminated tissue infection. An important diagnostic and prognostic factor for the treatment of infection is a quick and correct diagnosis of amoebae strains. The aim of our study was to develop a rapid method for detection and identification of pathogenic Acanthamoeba spp. strains from diagnostic material collected from water. In this study we analysed five amplification-based genetic markers (Aca 16S, Ac6/210, GP, JDP, Nelson) used for identification of pathogenic Acanthamoeba spp. strains isolated in water sources in Poland, Iceland and Sweden. Our results demonstrated the presence of pathogenic Acanthamoeba strains in tap water. PCR assay appeared to be a more rapid and sensitive method to detect the presence of amoebae than the limited conventional techniques. Based on our observations, we can confirm that the use of four out of five genetic markers (Aca 16S, Ac 6/210, JDP, GP, Nelson) may be helpful in identification of Acanthamoeba spp. strains, but only one Aca 16S primer pair is a highly specific marker that distinguishes between pathogenic strains of Acanthamoeba and other free-living amoeba families.

The use of phytotherapy in diseases caused by parasitic protozoa.

The paper presents an overview of the use of natural therapeutic agents in combating parasitic diseases. Nowadays there is increasing demand for proven plant therapies, which often are found to be more effective than synthetic pharmaceuticals in chronic diseases. In many cases herbal preparations perfectly supplement the conventional treatment and at the same time do not cause side effects. On the pharmaceutical market there are many drugs of plant origin which have been applied in the treatment of parasitic diseases. However, researchers are still looking for new plants, or specific substances isolated from them, which can be used in therapy. In this paper, drugs of plant origin used in the treatment of amoebiasis, giardiasis, malaria, leishmaniasis, trypanosomiasis and acanthamoebiasis are described.

[Use of the presence of cellulose in cellular wall of Acanthamoeba cysts for diagnostic purposes]. / Wykorzystanie obecnosci celulozy w scianie komórkowej cyst Acanthamoeba do celów diagnostycznych.

Species identification within the genus Acanthamoeba is based predominantly on morphological and biochemical features. It is labor-intensive and requires cloning and axenization. We described a novel immunocytochemical method for the identification of Acanthamoeba spp. based on selective binding of Clostridium cellulovorans cellulase to protozoan cyst wall cellulose. Free-living amoebae isolated from different water sources by filtration and subsequent cultivation on non-nutrient agar were assigned to genera Acanthamoeba, Naegleria or Hartmannella using morphological taxonomic criteria. Tissues samples from experimentally infected mice were fixed in formalin and for sectioning embedded in paraffin or snap frozen. The Cellulose-Binding Domain of C. cellulovorans cellulase (CBD) obtained as a recombinant protein, were coupled to the fluorescent dye using Alexa Fluor350, 488, 568 - Protein Labelling Kit or labelled with the biotin using EZ-Link Sulfo-NHS-Biotin. All coupling procedures were performed according to the methods provided by manufacturers. For staining with CBD conjugate, slides containing cysts collected from the agar plates or tissue sections were immersed with PBS and incubated with CBD for 30 min at room temperature, washed 3 times with PBS. For staining with CBD-biotin slides containing cysts were incubated with biotinylated CBD for 30 min at room temperature. Subsequent washings in changes of PBS were followed by the incubation with Strept ABComplex/HRP, for 30 min at room temperature, than 3,3 diaminobenzidine tetrahydrochloride was added for 15 min. Slides were rinsed with water, dried and examined in the light microscope. We showed that cellulose could be easily detected by immunofluorescence using conjugated CBD in the inner cyst wall of Acanthamoeba spp. The reference strains of Acanthamoeba spp. and all Acanthamoeba strains isolated from water and from tissues of infected animals gave positive reaction. CBD prepared as a biotynylated protein can be also used for the demonstration of Acanthamoeba cyst in infected tissues and environmental samples.