The wild boar (Sus scrofa) population has increased dramatically over the last decades throughout Europe and it has become a serious pest. In addition, the common habitat of wild boar and of the tick, Ixodes ricinus, indicates the potential of wild boar to play a role in epidemiology of epizootic and zoonotic tick-borne pathogens, including Anaplasma phagocytophilum. In Europe, epidemiological cycles and reservoirs of A. phagocytophilum, including its zoonotic haplotypes, are poorly understood. In this study, we focused on detection and further genetic characterization of A. phagocytophilum and piroplasmids in 550 wild boars from eleven districts of Moravia and Silesia in the Czech Republic. Using highly sensitive nested PCR targeting the groEL gene, the DNA of A. phagocytophilum was detected in 28 wild boars (5.1 %) representing six unique haplotypes. The dominant haplotype was found in 21 samples from 7 different districts. All detected haplotypes clustered in the largest clade representing the European ecotype I and the dominant haplotype fell to the subclade with the European human cases and strains from dogs and horses. Nested PCR targeting the variable region of the 18S rRNA gene of piroplasmids resulted in one positive sample with 99.8 % sequence identity to Babesia divergens. The presence of these two pathogens that are primarily circulated by I. ricinus confirms the local participation of wild boar in the host spectrum of this tick and warrants experimental studies to address wild boar as a reservoir of zoonotic haplotypes of A. phagocytophilum.

• MeSH
• Anaplasma phagocytophilum genetika   izolace a purifikace   MeSH
• anaplasmóza epidemiologie   mikrobiologie   MeSH
• babezióza epidemiologie   parazitologie   MeSH
• bakteriální geny MeSH
• genetická variace * MeSH
• nemoci prasat epidemiologie   mikrobiologie   parazitologie   MeSH
• Piroplasmida genetika   izolace a purifikace   MeSH
• prasata MeSH
• prevalence MeSH
• protozoální geny MeSH
• Sus scrofa MeSH
• zdroje nemoci parazitologie   veterinární   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Myxozoan parasites of the genus Kudoa Meglitsch, 1947 are associated with post-mortem tissue degradation that causes great financial losses to commercial fisheries. Kudoa thyrsites (Gilchrist, 1924) is a species with a very wide host range including commercial tunas, mackerels, salmonids and flatfishes. A sample of 190 fishes of 18 species from the Madeira Archipelago and 30 Atlantic chub mackerel, Scomber colias Gmelin, and 30 blue whiting, Micromesistius poutassou (Risso), from the Portuguese mainland coast were examined for the presence of species of Kudoa. The prevalence of Kudoa spp. was 80% in M. poutassou and 60% in S. colias. No spore was detected in S. colias from Madeira, which was confirmed by specific PCR screening of the muscle from all individuals of S. colias. SSU rDNA analysis revealed that M. poutassou and S. colias from the Portuguese mainland coast were infected with K. thyrsites, an economically important myxozoan parasite. Both sequences were identical with sequences of the eastern Atlantic K. thyrsites genotype, including that from the type host of this parasite. This is the first report of K. thyrsites from M. poutassou and S. colias. The fact that spores of species of Kudoa were not detected in fishes screened in the Madeira Archipelago may be explained by various ecological factors, such as the absence of a continental shelf, a short insular shelf, and oceanic waters with low productivity, all resulting in reduced abundance of benthic organisms. Consequently, it is possible that as yet unknown annelid definitive hosts of Kudoa spp. are absent or very rare near Madeiran coasts.

• MeSH
• fylogeneze MeSH
• Gadiformes parazitologie   MeSH
• Myxozoa * klasifikace   genetika   izolace a purifikace   MeSH
• nemoci ryb parazitologie   MeSH
• Perciformes parazitologie   MeSH
• prevalence MeSH
• protozoální geny MeSH
• ribozomální DNA genetika   MeSH
• ryby parazitologie   MeSH
• spory cytologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Portugalsko MeSH

Parasites of the genus Cryptosporidium Tyzzer, 1910 are one of the most common protistan parasites of vertebrates. Faecal samples from 179 red foxes (Vulpes vulpes [Linnaeus]), 100 grey wolves (Canis lupus Linnaeus), 11 golden jackals (Canis aureus Linnaeus), and 63 brown bears (Ursus arctos Linnaeus) were collected in the Czech Republic, Poland and Slovakia. Samples were examined for the presence of Cryptosporidium spp. using microscopy and PCR/sequence analysis. Phylogenetic analysis based on the small subunit ribosomal RNA (SSU), actin and 60-kDa glycoprotein (gp60) genes using the maximum likelihood method revealed the presence of Cryptosporidium tyzzeri Ren, Zhao, Zhang, Ning, Jian et al., 2012 (n = 1) and C. andersoni Lindsay, Upton, Owens, Morgan, Mead et Blackburn, 2000 (n = 2) in red foxes, C. canis Fayer, Trout, Xiao, Morgan, Lai et Dubey, 2001 (n = 2) and C. ubiquitum Fayer, Santín et Macarisin, 2010 (n = 2) in grey wolves, and C. galli Pavlásek, 1999 in brown bears (n = 1) and red foxes (n = 1). Subtyping of isolates of C. ubiquitum and C. tyzzeri based on sequence analysis of gp60 showed that they belong to the XIId and IXa families, respectively. The presence of specific DNA of C. tyzzeri, C. andersoni and C. galli, which primarily infect the prey of carnivores, is probably the result of their passage through the gastrointestinal tract of the carnivores. Finding C. ubiquitum XIId in wolves may mean broadening the host spectrum of this subtype, but it remains possible this is the result of infected prey passing through the wolf - in this case deer, which is a common host of this parasite. The dog genotype of C. canis was reported for the first time in wolves.

• MeSH
• Carnivora parazitologie   MeSH
• Cryptosporidium * genetika   izolace a purifikace   MeSH
• feces parazitologie   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genotypizační techniky MeSH
• kryptosporidióza epidemiologie   MeSH
• lišky parazitologie   MeSH
• malé podjednotky ribozomu genetika   MeSH
• medvědovití parazitologie   MeSH
• prevalence MeSH
• protozoální DNA genetika   MeSH
• protozoální geny MeSH
• psi parazitologie   MeSH
• šakali parazitologie   MeSH
• vlci parazitologie   MeSH
• zvířata MeSH
• Check Tag
• psi parazitologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Evropa MeSH
• Polsko MeSH
• Slovenská republika MeSH

INTRODUCTION: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). METHODS: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. RESULTS: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. DISCUSSION: The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. CONCLUSIONS: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites.

• MeSH
• diferenciální diagnóza MeSH
• feces parazitologie   MeSH
• kočky parazitologie   MeSH
• kokcidióza veterinární   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• molekulární patologie metody   MeSH
• myši parazitologie   MeSH
• oocysty genetika   izolace a purifikace   MeSH
• protozoální geny MeSH
• repetitivní sekvence nukleových kyselin MeSH
• Sarcocystidae * genetika   izolace a purifikace   MeSH
• Toxoplasma * genetika   izolace a purifikace   MeSH
• toxoplazmóza zvířat MeSH
• zvířata MeSH
• Check Tag
• kočky parazitologie   MeSH
• myši parazitologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Cryptosporidium parvum is a zoonotic pathogen worldwide. Extensive genetic diversity and complex population structures exist in C. parvum in different geographical regions and hosts. Unlike the IIa subtype family, which is responsible for most zoonotic C. parvum infections in industrialized countries, IId is identified as the dominant subtype family in farm animals, rodents and humans in China. Thus far, the population genetic characteristics of IId subtypes in calves in China are not clear. METHODS: In the present study, 46 C. parvum isolates from dairy and beef cattle in six provinces and regions in China were characterized using sequence analysis of eight genetic loci, including msc6-7, rpgr, msc6-5, dz-hrgp, chom3t, hsp70, mucin1 and gp60. They belonged to three IId subtypes in the gp60 gene, including IIdA20G1 (n = 17), IIdA19G1 (n = 24) and IIdA15G1 (n = 5). The data generated were analyzed for population genetic structures of C. parvum using DnaSP and LIAN and subpopulation structures using STRUCTURE, RAxML, Arlequin, GENALEX and Network. RESULTS: Seventeen multilocus genotypes were identified. The results of linkage disequilibrium analysis indicated the presence of an epidemic genetic structure in the C. parvum IId population. When isolates of various geographical areas were treated as individual subpopulations, maximum likelihood inference of phylogeny, pairwise genetic distance analysis, substructure analysis, principal components analysis and network analysis all provided evidence for geographical segregation of subpopulations in Heilongjiang, Hebei and Xinjiang. In contrast, isolates from Guangdong, Shanghai and Jiangsu were genetically similar to each other. CONCLUSIONS: Data from the multilocus analysis have revealed a much higher genetic diversity of C. parvum than gp60 sequence analysis. Despite an epidemic population structure, there is an apparent geographical segregation in C. parvum subpopulations within China.

• MeSH
• Cryptosporidium parvum genetika   izolace a purifikace   MeSH
• Cryptosporidium genetika   izolace a purifikace   MeSH
• fylogeneze MeSH
• fylogeografie MeSH
• genetická variace MeSH
• genotyp MeSH
• kryptosporidióza epidemiologie   MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• nemoci skotu epidemiologie   parazitologie   MeSH
• populační genetika MeSH
• protozoální geny MeSH
• skot MeSH
• zoonózy epidemiologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Čína MeSH

Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.

• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• dyneiny biosyntéza   MeSH
• exprese genu MeSH
• protozoální geny MeSH
• protozoální proteiny biosyntéza   izolace a purifikace   MeSH
• rekombinantní proteiny biosyntéza   izolace a purifikace   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Avian cryptosporidiosis is a common parasitic disease that is caused by five species, which are well characterised at the molecular and biological level, and more than 18 genotypes for which we have limited information. In this study, we determined the occurrence and molecular characteristics of Cryptosporidium spp. in farmed ostriches in the Czech Republic. METHODS: The occurrence and genetic identity of Cryptosporidium spp. were analysed by microscopy and PCR/sequencing of the small subunit rRNA, actin, HSP70 and gp60 genes. Cryptosporidium avian genotype II was examined from naturally and experimentally infected hosts and measured using differential interference contrast. The localisation of the life-cycle stages was studied by electron microscopy and histologically. Infectivity of Cryptosporidium avian genotype II for cockatiels (Nymphicus hollandicus (Kerr)), chickens (Gallus gallus f. domestica (L.)), geese (Anser anser f. domestica (L.)), SCID and BALB/c mice (Mus musculus L.) was verified. RESULTS: A total of 204 individual faecal samples were examined for Cryptosporidium spp. using differential staining and PCR/sequencing. Phylogenetic analysis of small subunit rRNA, actin, HSP70 and gp60 gene sequences showed the presence of Cryptosporidium avian genotype II (n = 7) and C. ubiquitum Fayer, Santín & Macarisin, 2010 IXa (n = 5). Only ostriches infected with Cryptosporidium avian genotype II shed oocysts that were detectable by microscopy. Oocysts were purified from a pooled sample of four birds, characterised morphometrically and used in experimental infections to determine biological characteristics. Oocysts of Cryptosporidium avian genotype II measure on average 6.13 × 5.15 μm, and are indistinguishable by size from C. baileyi Current, Upton & Haynes, 1986 and C. avium Holubová, Sak, Horčičková, Hlásková, Květoňová, Menchaca, McEvoy & Kváč, 2016. Cryptosporidium avian genotype II was experimentally infectious for geese, chickens and cockatiels, with a prepatent period of four, seven and eight days post-infection, respectively. The infection intensity ranged from 1000 to 16,000 oocysts per gram. None of the naturally or experimentally infected birds developed clinical signs in the present study. CONCLUSIONS: The molecular and biological characteristics of Cryptosporidium avian genotype II, described here, support the establishment of a new species, Cryptosporidium ornithophilus n. sp.

• MeSH
• Cryptosporidium klasifikace   genetika   ultrastruktura   MeSH
• fylogeneze MeSH
• hospodářská zvířata parazitologie   MeSH
• hostitelská specificita MeSH
• klasifikace MeSH
• kryptosporidióza parazitologie   MeSH
• nemoci ptáků parazitologie   MeSH
• protozoální geny genetika   MeSH
• ptáci parazitologie   MeSH
• stadia vývoje MeSH
• Struthioniformes parazitologie   MeSH
• taxonomické DNA čárové kódování veterinární   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Apicomplexan parasites of the genus Sarcocystis have an obligate two-host life-cycle and comprise about 200 species, which infect different cold- and warm-blooded hosts, including humans. Recently, morphological and molecular studies of sarcocysts in broadly spread carnivore hosts have been on the rise. The description of muscular tissues infection by Sarcocystis in the raccoon dog and the common raccoon from the Czech Republic is herein presented. METHODS: During January-August 2019, 15 raccoon dogs and 1 common raccoon were examined from 5 districts (Česká Lípa, Liberec, Mladá Boleslav, Trutnov and Ústí nad Labem) of the Czech Republic. Muscle parts (diaphragm, forearm, hind-limb, tongue and heart) were examined in wet preparations under compression by light microscopy. After finding Sarcocystis sp., morphological characteristics and molecular analyses of 18S rRNA, 28S rRNA, ITS1 and cox1 loci were used to identify the species. RESULTS: Sarcocysts were detected and identified in 1 out of 15 raccoon dogs and in the single common raccoon. Preferential infection sites were diaphragm and tongue, followed by forearm and hind limb. To our knowledge, this is the first identification of microscopic sarcocysts by multi-locus genetic analysis from both host species. Molecular analyses revealed 100% similarity at 18S rRNA, 28S rRNA, and cox1 genes with S. lutrae for both hosts and 98-100% identity at the ITS1 region of the isolate from the common raccoon. CONCLUSIONS: Both widely distributed non-indigenous wild carnivores represent new intermediate host records for S. lutrae and the first report of this parasite in a member of the family Procyonidae, but still with an unknown natural definitive host. Molecular data revealed that this parasite species appears more closely related to the Sarcocystis spp. using raptorial birds as definitive hosts. Therefore, further studies aimed at its identification, including the complete life-cycle, remain necessary.

• MeSH
• fylogeneze MeSH
• molekulární patologie MeSH
• mývalové parazitologie   MeSH
• protozoální geny MeSH
• protozoální infekce MeSH
• psík mývalovitý parazitologie   MeSH
• RNA ribozomální 18S genetika   MeSH
• RNA ribozomální 28S genetika   MeSH
• Sarcocystidae klasifikace   MeSH
• Sarcocystis klasifikace   genetika   izolace a purifikace   MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

OBJECTIVES: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. METHODS: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. RESULTS: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. CONCLUSION: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.

• MeSH
• antiprotozoální látky farmakologie   MeSH
• fosforylcholin analogy a deriváty   farmakologie   MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania infantum * účinky léků   genetika   patogenita   MeSH
• leishmanióza viscerální farmakoterapie   patologie   MeSH
• léková rezistence genetika   MeSH
• membránové transportní proteiny genetika   MeSH
• myši inbrední BALB C parazitologie   MeSH
• myši MeSH
• Phlebotomus parazitologie   MeSH
• protozoální geny MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 μm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 μm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.

• MeSH
• Isospora klasifikace   cytologie   genetika   růst a vývoj   MeSH
• izosporóza parazitologie   veterinární   MeSH
• oocysty klasifikace   cytologie   genetika   růst a vývoj   MeSH
• Passeriformes parazitologie   MeSH
• protozoální geny genetika   MeSH
• sporozoiti klasifikace   cytologie   genetika   růst a vývoj   MeSH
• střeva parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH