Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Detection of Cyclospora cayetanensis, Echinococcus multilocularis, Toxocara spp. and microsporidia in fresh produce using molecular methods: - A review.

The current trend for a healthy lifestyle corresponds with a healthy diet, which is associated with regular and frequent consumption of raw fruit and vegetables. However, consumption of ready-to-eat (RTE) food without heat treatment or sufficient washing may pose a risk to consumers. Among the well-known protozoan parasites associated with RTE food and water are Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii. These belong among prioritized parasitic pathogens, as they are associated with numerous disease outbreaks in humans all around the world. Nevertheless, other parasitic agents such as Cyclospora cayetanensis, Toxocara cati, Toxocara canis, Echinococcus multilocularis and zoonotic microsporidia should not be neglected. Although these selected parasites belong to phylogenetically diverse groups, they have common characteristics associated with fresh produce and each of them poses a health risk to humans. Ensuring healthy food is produced requires the standartization of laboratory methods for the detection of parasitic agents. This article reviews the molecular methods currently used in laboratories for detection of Cyclospora cayetanensis, Toxocara cati, Toxocara canis, Echinococcus multilocularis and zoonotic microsporidia in fresh produce.

First autochtonous infection of a dog with Oslerus (Filaroides) osleri in the Czech Republic.

This case report describes an infection with O. osleri in a 10-month-old intact female Miniature German Spitz that presented with a 3-month history of progressive cough. Diagnosis was based upon visualization of characteristic lesions during bronchoscopy. Female parasites and first-stage larvae collected from tracheal nodules were morphologically identical to the larvae of O. osleri. First-stage larvae isolated from faeces were used for morphological and molecular confirmation of the diagnosis. Anthelmintic therapy with fenbendazole (50 mg/kg orally once daily for 2 weeks) was successful. This is the first report of autochthonous infection of a dog with O. osleriin the Czech Republic. Oslerosis should be considered in the differential diagnosis in young dogs with persistent respiratory signs.

Enterocytozoon bieneusi in Bovine Viral Diarrhea Virus (BVDV) infected and noninfected cattle herds.

Enterocytozoon bieneusi known as a causative agent of opportunistic infections instigating diarrhoea in AIDS patients was identified also in a number of immunocompetent patients and in a wide range of animals, including cattle. In the present study we tested if the Bovine Viral Diarrhea Virus (BVDV), the most common pathogen underlying immunosuppressive Bovine Viral Diarrhoea (BVD), can enhance the occurrence of opportunistic infections with E. bieneusi in cattle. Six dairy farms were investigated using ELISA to detect antibodies against or antigens arising from BVDV in collected sera. A total of 240 individual faecal samples from four age groups were examined for the presence of E. bieneusi by nested PCR. Sequence analysis of six E. bieneusi positive samples revealed the presence of the genotype I of E. bieneusi, previously described in cattle. The hypothesis expecting higher prevalence of E. bieneusi in BVDV positive cattle herds was not confirmed in this study; however this is the first description about E. bieneusi in cattle in the Czech Republic.

The comparison of different ELISA procedures in detecting anti-Trichinella IgG in human infections.

The aim of this study was to compare the performance of four different ELISA procedures in diagnosing Trichinella spp. infections in human outbreaks. The main differences between the various procedures were: antigen concentration, dilutions of human serum sample, the type of conjugate used, and time of conjugate incubation. Serum samples were collected from 23 individuals involved in an outbreak. Results obtained in the four tested ELISA procedures differed in the rate of positive results obtained for the examined outbreak. Western blot confirmed the ELISA-positive results. Reactions to the specific 41-45kDa and 55kDa bands were obtained with all positive sera.

Prevalence and intensity of infections in the lymnaeid snail Omphiscola glabra experimentally infected with Fasciola hepatica, Fascioloides magna and Paramphistomum daubneyi.

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20 degrees C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6-8.3% instead of 23.6-25.9%), the total number of free rediae (10.7-17.9 per snail instead of 26.3-34.7), and that of free cercariae (112.8-136.9 per snail instead of 177.8-248.5). Despite a greater number of free rediae at day 75 p.e. (36.2-45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.

Serological survey of Neospora caninum in dogs in the Czech Republic and a long-term study of dynamics of antibodies.

The main aim of the present study was to establish the prevalence of antibodies against Neospora caninum dogs from the Czech Republic and to examine the dynamics of antibody titers during a long-term period. For this purpose, sera of 858 dogs were examined for the presence of anti-N. caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Four groups of dogs of various origins were included in the survey: the first group (A, n=470) comprised dogs purchased by the Czech Army from the civilian sector throughout the Czech Republic, with 22 (4.7%) N. caninum-positive dogs, second group (B, n=115) represented police dogs with no seropositive animal, third group (C, n=195) were pet dog sera collected for veterinary clinic with 5 (2.6%) anti-N. caninum sera and the fourth group (D, n=78) of canine shelter dogs with the seroprevalence of 19.2%. The differences in seroprevalence were significant (P< or =0.01) between groups B and A, and between D and A. None of the serologically positive animals had clinical signs of neurological disorders. Coprological examination did not reveal any dog shedding N. caninum oocysts. The seropositivity rates for N. caninum were analyzed in relation to other data, such as age, breed and gender. Increased prevalence rates of anti-N. caninum antibodies were found in the older age strata of the dog population sample tested in the present study. We found significantly higher (P=0.02) prevalence in 3-3.5-year-old dogs (11.1% of 36), as compared to 1-1.5-years-old dogs (2% of 98). A longitudinal study of antibody dynamics was carried out in 19 initially seropositive dogs over a period of 4 years. The second and third examinations revealed that antibody titers decreased in majority of positive dogs (10, 52.6%), of which in seven cases (36.8%) the titers fell to levels that are currently considered as being seronegative (titer <1:50), or even became undetectable (titer <1:25).

Effects of low and high temperatures on viability of Parascaris equorum eggs suspended in water.

Microcentrifuge tubes containing 5000 eggs of Parascaris equorum suspended in water were frozen at -5, -10, -15, -20, and -80 degrees C for 1-168 h and then thawed at a room temperature. Other samples of P. equorum eggs suspended in water were inserted into wells in the heated metal block of a thermal DNA cycler. Block temperatures were set at 5 degrees C incremental temperatures from 40 to 100 degrees C. At each temperature setting microcentrifuge tubes containing P. equorum eggs were removed 1 and 5 min later. Both, frozen and heated egg suspensions as well as untreated control suspensions were then incubated to test of viability based on the development of infective larvae inside viable eggs. We found out that eggs of P. equorum in water can retain viability and infectivity after freezing and that eggs survive longer at higher freezing temperatures. Our results also indicated that when water containing P. equorum eggs reached temperatures of 60 degrees C or higher within 1 min, the viability of eggs was lost.

Cercarial production of Fascioloides magna in the snail Galba truncatula (Gastropoda: Lymnaeidae).

Single-miracidium infections of Fascioloides magna in two populations of Galba truncatula were carried out under laboratory conditions to count free rediae and cercariae in snail cadavers just after death. Cercaria-shedding snails were in low numbers, and their shell height at day 60 p.e. was significantly greater than that of numerous infected snails that died without cercarial shedding. In snails that died between days 44 and 60 p.e. (at 20 degrees C), the numbers of second-generation rediae significantly increased with increasing shell heights of infected snails. First-generation rediae showed insignificant, quantitative variations, while scarce rediae of the third generation were only found in the highest snails. Cercariae were only produced by the second redial generation. In both groups of snails, free cercariae appeared from 6 mm of shell height, and their numbers increased in the upper classes up to 32.9 per snail. Metacercariae were only found from 9 mm of shell height and were in low numbers. The global cercarial production ranged from 163.5 to 210.0 in the highest classes of snail size from both groups and was limited, whereas the mean burdens of free rediae fluctuated from 39.5 to 43.9. The death of numerous infected snails without cercarial shedding might be explained by the presence of a very high number of second-generation rediae simultaneously growing within the body of these snails.

First studies on the susceptibility of Omphiscola glabra (Gastropoda: Lymnaeidae) from central France to Fascioloides magna.

Experimental infections of Omphiscola glabra (preadult snails), originating from central France, to a Czech isolate of Fascioloides magna miracidia were carried out to determine if the local populations of O. glabra may ensure the larval development of this parasite and to compare these results with those noted for a natural snail host, Galba truncatula. The presence of experimentally infected snails was noted in the six populations of snails studied. However, only a few snails shed their cercariae (O. glabra 5.3 to 17.1%, G. truncatula 15.1% in the first population, and no shedding in the other). The shell heights of cercariae-shedding (CS) snails were significantly greater than those of other infected snails, for O. glabra as well as for G. truncatula. The number of metacercariae noted in each snail group was low and showed insignificant variations. When experimental infections of O. glabra were performed in relation to the shell height of snails (from 1 to 14 mm) at miracidial exposure, the prevalence of infected snails significantly decreased with increasing shell heights at exposure. However, the presence of CS snails was only noted from the 5-6 to the 9-10 mm groups, and the mean number of metacercariae per group ranged from 27 to 44.2. Despite the high infectivity of the Czech isolate of F. magna miracidia, there was an incomplete adaptation with the French G. truncatula and O. glabra used in this study, as the metacercarial production was low, and cercarial shedding only occurred for snails which showed a strong increase of their shell height during F. magna infections.

Stimulation of innate immunity in newborn kids against Cryptosporidium parvum infection-challenge by intranasal/per-oral administration of liposomal formulation of N-L18-norAbu-GMDP adjuvant.

The effects of a liposomal preparation of lipophilic immunomodulator beta-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance to Cryptosporidium parvum infection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 microg, 200 microg, or 1000 microg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1 x 10(7) oocysts of C. parvum in 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 microg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P < 0.05). No cryptosporidia were found on the mucosal surface of treated kids by day 10, while the intestines of the control kids were still infected. All doses and routes of administration were judged effective with respect to suppression of cryptosporidia infections.

Tetratrichomonads from the oral cavity and respiratory tract of humans.

To clarify the taxonomy of trichomonads associated with human respiratory diseases, we examined a collection of axenic trichomonad strains isolated from the oral cavity and bronchi of patients from pulmonary diseases clinics in Tallin, Estonia. The oral and bronchial strains were compared mutually as well as with a reference strain of Trichomonas tenax, a common inhabitant of the human oral cavity, and other trichomonad species from humans and animals. Unexpectedly, the morphological studies, as well as DNA sequencing of ITS1-5.8S rRNA-ITS2 regions revealed that the Estonian strains belong to the genus Tetratrichomonas, with a high similarity to the avian species Tetratrichomonas gallinarum. None of the strains belonged to Trichomonas tenax. DNA fingerprinting using the RAPD method separated Estonian strains into 2 distinct groups: 'bronchial' consisting of 5 and 2 strains isolated from bronchi and 'oral' cavity, respectively, and oral consisting of 3 oral strains. Consistent differences between 'bronchial' and 'oral' groups were confirmed by analysis of ITS1-5.8S rRNA-ITS2 sequences. Our results have revealed novel trichomonad species of the human oral cavity and bronchi.

Experimental infection of severe combined immunodeficient (SCID) mice with the human microsporidian Trachipleistophora hominis.

Different courses of microsporidiosis, related to the route of infection, were observed in severe combined immunodeficient (SCID) mice inoculated with spores of the human microsporidian Trachipleistophora hominis (Phylum Microspora). After eye contamination by spores the mice became moribund within 7 to 8 weeks, showing severe infection in the conjunctiva and cornea, and lighter infections in the urinary bladder, liver and spleen. The mean survival time of intramuscularly inoculated SCID mice was 12 weeks, when heavy infection was found in muscles around the site of inoculation, and also in several viscera. Subcutaneously inoculated SCID mice developed skin lesions around the inoculation sites, and heavy urinary bladder infection, and died 6 or 7 weeks after inoculation. Intracerebrally inoculated SCID mice became moribund 5 or 6 weeks after inoculation with massive infection in the urinary bladder and liver, but none in the brain. Intraperitoneally inoculated SCID mice survived for 13 weeks and the urinary bladder and liver were the most heavily infected organs. The SCID mice, inoculated perorally and examined after 23 weeks, were uninfected. Infection was not detected in the brain of any of the inoculated SCID mice. Our results show that T. hominis has very little tissue specificity. Peroral infection seems to be ineffective in T. hominis, but eye conta mination or insect bite (as mimicked by injection) are suggested as possible routes of infection under natural conditions.

Seroprevalence of Neospora caninum in aborting dairy cattle in the Czech Republic.

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.

Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum.

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Caryospora matatu n. sp., a new coccidian parasite (Apicomplexa: Eimeriidae) from the horned bush-viper, Atheris ceratophorus Werner, 1895 from Tanzania.

Caryospora matatu n. sp. is the first species of coccidia reported from the a horned bush viper, Atheris ceratophorus endemic to Tanzania. Oocysts are spherical or slightly subspherical, 19.8 (16-23) microns, a micropyle and an oocyst residuum are absent, a single polar granule is present. An oocyst wall is bilayered, approximately 1.5 microns thick, brownish and distinctly pitted. Sporocysts are ellipsoidal, 15.6 (12.5-17.0) x 10.1 (8-12) microns, tightly fitting an internal oocyst diameter, Stieda and substieda bodies are present. An experimental inoculation of SCID mice did not confirm a facultatively heteroxenous life cycle in the studied coccidium. Morphology and evolutionary history of Caryospora species from viperids is is reviewed and discussed.

Eimeria hajeki n. sp. (Apicomplexa: Eimeriidae), a new coccidian parasite of the pygmy chameleon, Rampholeon temporalis (Matschie, 1892) (Reptilia: Chamaeleonidae) from Usambara Mountains, Tanzania.

Fecal samples from 10 pygmy chameleons, Rampholeon temporalis (Matschie, 1892), an endemic species of the Usambara Mountains in northeastern Tanzania, were examined for coccidian parasites. Two (20%) chameleons were found to be passing oocysts of Eimerio Schneider. Comparison with other species of Eimeria indicates that the coccidian found represents a new species. Sporulated oocysts of Eimeria hajeki n. sp. are oval, 30.2 (29-31) by 23.5 (22-25) microm, with a shape index (length/width) of 1.3 (1.2-1.4) and a 2-microm-thick rough, bilayered wall. Micropyle and polar granule are absent. Sporocysts are oval to rhomboidal, 10.8 (9-11.5) by 8.8 (7.5-10) microm, with a shape index of 1.2 (1.15-1.3) and a wall composed of 2 valves joined by a suture.

The human isolate of Brachiola algerae (Phylum Microspora): development in SCID mice and description of its fine structure features.

Ocular, peroral, intraperitoneal, intramuscular, and subcutaneous inoculation of severe combined immunodeficient (SCID) mice with spores of the human isolate (CDC: V404) of Brachiola algerae (syn. Nosema algerae) (Phylum Microspora) revealed that the microsporidium develops in viscera of the immunodeficient mouse host, but only after the ocular administration of spores. It is hypothesized that the physico-chemical milieu of the conjunctiva and cornea helped to adapt the originally 'poikilothermic microsporidian' to the conditions within the homoiothermic organism. Ocular application of spores caused no clinical signs of disease at the application site. However, severe infection in the liver was found 60 days after infection, manifested as hepatosplenomegaly and multifocal miliary necroses and granulomas containing parasites. No microsporidia were found in any other tissues. Transmission electron microscopy revealed characteristic tubulovesicular 'secretory materials' on the plasma membrane of all developmental stages of B. algerae except sporoblasts and spores. These formations increase the parasite surface and allow more efficient metabolic communication of the parasite with the host cell. It is hypothesized that the presence of these structures is a factor helping the parasite to grow in a variety of hosts and tissues. Ultrastructural characters support the likelihood that B. algerae and B. vesicularum are conspecific, and that there exists a relationship between species of the genera Brachiola and Anncaliia.

Phylogenetic position of a renal coccidium of the European green frogs, 'Isospora' lieberkuehni Labbé, 1894 (Apicomplexa: Sarcocystidae) and its taxonomic implications.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.