A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

In vitro assay to determine inactivation of Toxoplasma gondii in meat samples.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 µL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Optimization of the most widely used serological tests for a harmonized diagnosis of Toxoplasma gondii infection in domestic pigs.

The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63-0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30-0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results.

Very low intraspecific sequence variation in selected nuclear and mitochondrial Parascaris univalens genes.

Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or the other species. Recently, only P. univalens has been identified by karyotyping and the last published study identifying P. equorum dates back to 1989. In order to improve species-specific detection, molecular markers are required. Here, partial 12S rRNA, cytochrome oxidase I (COI) and complete internal transcribed spacer (ITS)-1 and - 2 sequences were obtained from 24 karyotyped Parascaris specimens from Poland and 6 German specimens (not karyotyped) and used in phylogenetic analyses with orthologous sequences from GenBank. All karyotyped specimens were identified as P. univalens. In the phylogenetic analysis, they formed very homogenous clusters for all target genes and in a multi-locus analysis. Within this cluster, almost all sequences from GenBank were also included, no matter if they had been assigned to P. univalens or P. equorum. However, a small number of P. univalens ITS and COI sequences originating from donkeys from a single farm in China formed a highly supported sister cluster suggesting that they might represent another Parascaris genotype or species. Our data also strongly suggest that nearly all ITS and COI sequences previously deposited in GenBank and assigned to P. equorum actually represent P. univalens. The fact that significantly different sequences can be found in Parascaris spp. suggests that PCR-based species diagnosis will be possible once molecular markers have been identified for P. equorum from karyotyped specimens.

A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii.

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.

Correction to: Epidemiology of taeniosis/cysticercosis in Europe, a systematic review: eastern Europe.

In the original article [1], the authors Dr Jasmin OMERAGIC and Dr Davor ALAGIC were erroneously omitted from the co-authors list.

Epidemiology of taeniosis/cysticercosis in Europe, a systematic review: eastern Europe.

BACKGROUND: Taenia solium and Taenia saginata are food-borne parasites of global importance. In eastern Europe only fragmented information is available on the epidemiology of these zoonotic parasites in humans and animal populations. In particular for T. solium, on-going transmission is suspected. The aim of this systematic review was to collect the available data and describe the current knowledge on the epidemiology of T. solium and T. saginata in eastern Europe. METHODS: Literature published in international databases from 1990 to 2017 was systematically reviewed. Furthermore, local sources and unpublished data from national databases were retrieved from local eastern European experts. The study area included 22 countries. RESULTS: Researchers from 18 out of the 22 countries provided data from local and unpublished sources, while no contacts could be established with researchers from Belarus, Kosovo, Malta and Ukraine. Taeniosis and human cysticercosis cases were reported in 14 and 15 out of the 22 countries, respectively. Estonia, the Former Yugoslav Republic of Macedonia, Lithuania, Moldova, Poland, Romania, Serbia, and Slovakia reported cases of porcine cysticercosis. Croatia, Czech Republic, Estonia, Former Yugoslav Republic of Macedonia, Moldova, Poland, Romania, Serbia, Slovakia, and Ukraine reported bovine cysticercosis. CONCLUSIONS: There is indication that taeniosis and cysticercosis are present across eastern Europe but information on the occurrence of T. solium and T. saginata across the region remains incomplete. Available data are scarce and species identification is in most cases absent. Given the public health impact of T. solium and the potential economic and trade implications due to T. saginata, notification of taeniosis and human cysticercosis should be implemented and surveillance and notification systems in animals should be improved.

Predilection sites for Toxoplasma gondii in sheep tissues revealed by magnetic capture and real-time PCR detection.

Undercooked lamb and mutton are common sources of Toxoplasma gondii infection for humans. A sequence specific magnetic capture technique in combination with quantitative real-time PCR targeting the 529 bp repeat element of T. gondii was used for estimation of the parasite burdens in various sheep tissues (n = 6) three months after peroral experimental inoculation with 10,000 T. gondii oocysts. Brain was the most frequently affected organ (positive in all 6 sheep) and showed the highest estimated parasite loads (0.5-30,913 parasites/g tissue). Lung samples were positive in three sheep, with load estimates of 36.3 to <1 parasite/g tissue. Heart tissue was positive in three sheep and kidney only in one animal with low parasite loads (<1 parasite/g tissue). Only few skeletal muscle samples in 2 animals showed positive results, with very low parasite burdens, while samples from further internal organs (i.e. liver and spleen) were negative in all animals. This study identified the brain as the most important predilection site and therefore the most appropriate tissue for T. gondii detection.

Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi in rabbits from different farming systems.

The breeding of domestic rabbit (Oryctolagus cuniculus) for human consumption has a long tradition mainly in European and Asian countries. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animals health. This study aimed to collect sera from rabbits bred in different conditions and test the presence of Toxoplasma gondii and Encephalitozoon cuniculi antibodies. Whether infections were active or latent was assessed by determining the occurrence of IgM or IgM together with IgG antibodies which indicated active infection whereas latent infection was characterized by finding IgG antibodies only. An ELISA test was performed with 1883 sera samples collected throughout the Czech and Slovak Republics. The seroprevalence of T. gondii in 902 samples from 6 commercial farms (CF) was very low with only 4 rabbits (0.4%) being positive. In total 99 (10.1%) individuals out of 981 samples from 29 household farms (HF) were positive for T. gondii antibodies. Only 2 (50%) of the T. gondii positive CF rabbits had active infections while the rest were latently infected. The serological results showed that 35 (35.4%) rabbits from the T. gondii positive HF group suffered from active infection. Out of CF samples 185 (20.5%) were positive for E. cuniculi. Antibodies of E. cuniculi were detected in 497 (50.7%) HF rabbits. Active E. cuniculi infections were determined in 85.9% of CF and 56.3% of HF rabbits; respectively. Interestingly, the E. cuniculi positive rabbits were significantly more often positive for anti-T. gondii antibodies in comparison to E. cuniculi negative individuals. Prevalence of T. gondii in CF rabbits was negligible. According to our results meat of HF rabbits still poses a risk of T. gondii infection. Nevertheless, the risk is on its lowest level in 20 years which is apparently caused due to changes in feeding practices. The occurrence of E. cuniculi antibodies was significantly lower in rabbits from commercial farms, apparently because of better hygiene conditions.

Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages.

Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of this study was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and in dry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity (aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentally infected animals was used for production of VP meat with or without addition of 2.5% curing salt, and stored at 4 °C or at -20 °C. Results of bioassay showed that, samples of vacuum packed Toxoplasma positive meat without salt addition were alive after six weeks at 4 °C. Incubation at -20 °C supported the viability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meat were still viable, but not after 14 days at 4 °C. All the DFS samples were not positive for infective cysts which mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuum packaging increases the survival of T. gondii cysts.

Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR.

Pigs represent an important source of food in many countries, and undercooked pork containing tissue cysts is one of the most common sources of Toxoplasma gondii infection for humans. A magnetic capture method for the isolation of T. gondii DNA and quantitative real-time PCR targeting the 529 bp TOXO repeat element were used to estimate the parasite burden in different tissues of pigs experimentally infected with T. gondii oocysts, and to determine the predilection sites of T. gondii in this host species. The highest concentration of T. gondii DNA was found in brain tissues, equivalent to [median] 553.7 (range 3857.7-121.9) parasites per gram, followed by lungs, heart and dorsal muscles with median values corresponding to 0.3 (range 61.3-0.02); 2.6 (range 7.34-0.37) and 0.6 (range 2.81-0.31) parasites per gram of tissue, respectively. Skeletal muscles from fore and hindlimb, liver and kidney presented very low infection burdens equivalent to [median] ≤0.2 parasites per gram of tissues, and no parasite DNA could be detected in the spleen. This study contributes to understanding the value of different pig tissues as a source of T. gondii infection for humans and shows that the brain, while not being of major importance as human food source, may represent a first-line selection tissue when performing non-serological surveys (e.g. bioassays, histopathological, immunohistochemical or molecular studies) to detect T. gondii infections in pigs.

Life cycle of Cryptosporidium muris in two rodents with different responses to parasitization.

This study focuses on mapping the life cycle of Cryptosporidium muris in two laboratory rodents; BALB/c mice and the southern multimammate rat Mastomys coucha, differing in their prepatent and patent periods. Both rodents were simultaneously experimentally inoculated with viable oocysts of C. muris (strain TS03). Animals were dissected and screened for the presence of the parasite using a combined morphological approach and nested PCR (SSU rRNA) at different times after inoculation. The occurrence of first developmental stages of C. muris in stomach was detected at 2.5 days post-infection (dpi). The presence of Type II merogony, appearing 36 h later than Type I merogony, was confirmed in both rodents. Oocysts exhibiting different size and thickness of their wall were observed from 5 dpi onwards in stomachs of both host models. The early phase of parasitization in BALB/c mice progressed rapidly, with a prepatent period of 7.5-10 days; whereas in M. coucha, the developmental stages of C. muris were first observed 12 h later in comparison with BALB/c mice and prepatent period was longer (18-21 days). Similarly, the patent periods of BALB/c mice and M. coucha differed considerably, i.e. 10-15 days vs chronic infection throughout the life of the host, respectively.

Assessment of diagnostic accuracy of a commercial ELISA for the detection of Toxoplasma gondii infection in pigs compared with IFAT, TgSAG1-ELISA and Western blot, using a Bayesian latent class approach.

Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs. In the absence of a readily available 'gold standard', an estimation of diagnostic accuracy is difficult to assess. A commercial ELISA (PrioCHECK® Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagnosis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustment for within indoor and outdoor farm clustering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-purified P30 (SAG1) T. gondii tachyzoite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n=149) and indoor (n=148) farms in Argentina. The estimated sensitivity and specificity for the commercial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n=6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2 weeks p.i. until the end of the observation period (11 weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK® Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagnosis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

[Monitoring of animal trichinellosis in the Czech Republic--the past and present]. / Monitoring trichinelózy zvírat v Ceské republice--historiea soucasnost.

The nematode Trichinella spp. is the etiological agent of trichinellosis, a zoonotic parasitic disease. Many carnivorous and omnivorous animal species may become infected with Trichinella spp., including humans. Trichinella spp. is transmitted orally through consumption of raw or undercooked meat. There is a general agreement that animals do not get sick following infection. However, the course of infection in humans includes disease that can range from subclinical to fatal. Because of its role in human disease, there are increasing global requirements for reliable diagnostic and control methods for Trichinella in food animals to ensure meat safety. This review article describes the biology and history of human and animal trichinellosis in the Czech Republic, and recommended test methods as well as modified and optimized procedures that are used in meat inspection programmes.

Opportunistic nature of the mammalian microsporidia: experimental transmission of Trachipleistophora extenrec (Fungi: Microsporidia) between mammalian and insect hosts.

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts (Vavraia culicis) or being able to infect both mammalian and insect host (Trachipleistophora hominis).

Development of a multilocus sequence tool for typing Cryptosporidium muris and Cryptosporidium andersoni.

Although widely used for the characterization of the transmission of intestinal Cryptosporidium spp., genotyping tools are not available for C. muris and C. andersoni, two of the most common gastric Cryptosporidium spp. infecting mammals. In this study, we screened the C. muris whole-genome sequencing data for microsatellite and minisatellite sequences. Among the 13 potential loci (6 microsatellite and 7 minisatellite loci) evaluated by PCR and DNA sequencing, 4 were eventually chosen. DNA sequence analyses of 27 C. muris and 17 C. andersoni DNA preparations showed the presence of 5 to 10 subtypes of C. muris and 1 to 4 subtypes of C. andersoni at each locus. Altogether, 11 C. muris and 7 C. andersoni multilocus sequence typing (MLST) subtypes were detected among the 16 C. muris and 12 C. andersoni specimens successfully sequenced at all four loci. In all analyses, the C. muris isolate (TS03) that originated from an East African mole rat differed significantly from other C. muris isolates, approaching the extent of genetic differences between C. muris and C. andersoni. Thus, an MLST technique was developed for the high-resolution typing of C. muris and C. andersoni. It should be useful for the characterization of the population genetics and transmission of gastric Cryptosporidium spp.

Usefulness of detection of specific IgM and IgG antibodies for diagnosis of clinical encephalitozoonosis in pet rabbits.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has wide host distribution, but primary affects rabbits. This study presents a seroepidemiological study of E. cuniculi infection in 500 pet rabbits from the Czech Republic using ELISA capable of measuring IgM and IgG antibodies. Specific IgM antibodies, reflecting acute, reactivated infection or reinfection, were detected in 32.4% of all rabbits. IgG antibodies indicating chronic infection, were presented in 68.0% of all rabbits. The highest detection rate of IgM (54.4%) and IgG (86.1%) antibodies was ascertained in rabbits with neurological symptoms (n=79, group I). In rabbits with renal disorders (n=47, group II) 36.2% animals were specific IgM and 80.9% IgG positive. Out of 9 rabbits with ocular disorders (group III), 44.4% were positive for anti-E. cuniculi IgM and 77.8% for IgG antibodies. In rabbits with multiple signs (neurological and renal or ocular, n=16, group IV), 43.8% animals were specific IgM and 68.8% IgG positive. Out of 287 rabbits with other disease (group V), 26.5% were positive for anti-E. cuniculi IgM and 64.1% for IgG antibodies. However, the high presence of IgM (24.2%) and IgG (51.6%) antibodies was detected in clinically healthy rabbits (n=62, group VI). Toxoplasma gondii infection should be considered as a differential diagnosis for neurological and ocular disorders in rabbits. Using ELISA, 19.2% from all rabbits were positive for specific anti-T. gondii IgG. The highest seropositivity was detected in group III (44.4%). Simultaneous testing of IgM and IgG specific antibodies give an indication of the infection status. Presence of IgM antibodies is indicative for active infection with requirement to institute proper antimicrosporidial therapy. As active infection was detected in considerably high numbers of rabbits with clinical signs that are not usually associated with E. cuniculi, and even in asymptomatic rabbits, detection of both isotypes of specific antibodies should be a routine part of a health check in rabbits.

Eugregarine trophozoite detachment from the host epithelium via epimerite retraction: fiction or fact?

Eugregarines represent a diverse group of Apicomplexa parasitising numerous invertebrates. Their sporozoites generally develop into epicellular trophozoites attached to the host epithelium by a specialised attachment organelle known as an epimerite. They are considered peculiar protists due to their unique cell architecture and dimensions as well as their attachment strategy which is similar to that of cryptosporidia. Using electron and fluorescence microscopy, the fine structure of the epimerite with associated structures and the mechanism of trophozoite detachment from the host epithelium were studied in Gregarina polymorpha parasitising the intestine of Tenebrio molitor larvae. The epimerite appears to be a very dynamic structure whose shape dramatically changes depending on whether or not it is embedded into the host epithelium. The trophozoite's most fragile zone is the area below the membrane fusion site at the epimerite base. The epimerite plasma membrane forms basal radial ribs which are involved in increasing its surface and strengthening the epimerite-host cell junction. FITC-phalloidin labelling demonstrated the presence of filamentous actin in trophozoites along with its accumulation at the epimerite base and in the apical end of the protomerite, as well as a patch accumulation of filamentous actin in the protomerite of maturing and mature trophozoites. Indirect immunofluorescence revealed the presence of myosin in the cortical zone of the epimerite and in the membrane fusion site area. The data obtained strongly suggest that these structures could facilitate the detachment of a mature trophozoite from the host epithelium. Supported by data presented herein and our previous observations, we propose a new hypothesis on the mechanism of trophozoite detachment from the host epithelium based on epimerite retraction into the protomerite. This is contrary to the commonly accepted hypothesis describing gradual epimerite constriction and subsequent separation facilitated by contractility of the membrane fusion site (osmiophilic ring).

New species of Cryptosporidium Tyzzer, 1907 (Apicomplexa) from amphibian host: morphology, biology and phylogeny.

Cryptosporidium fragile sp. n. (Apicomplexa) is described from black-spined toads, Duttaphrynus melanostictus (Schneider) (Amphibia, Anura, Bufonidae) from the Malay Peninsula. The parasitized animals were directly imported from Malaysia and harboured C. fragile at the time of arrival. Oocysts were subspherical to elliptical with irregular contour in optical section, measuring 6.2 (5.5-7.0) x 5.5 (5.0-6.5) microm. Oocyst wall was smooth and colourless in light microscopy. The endogenous development of C. fragile in the stomach of black-spined toad was analysed in detail using light and electron microscopy. Cryptosporidian developmental stages were confined to the surface of gastric epithelial cells. In transmission experiments, C. fragile has not been infective for one fish species, four amphibian species, one species of reptile and SCID mice. Full length small subunit rRNA gene sequence was obtained. Phylogenetic reconstruction revealed distinct status of C. fragile within the clade of species with gastric localisation including Cryptosporidium muris Tyzzer, 1907, Cryptosporidium serpentis Levine, 1980 and Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead et Blagburn, 2000. Described characteristics differentiate C. fragile from the currently recognized Cryptosporidium species. Our experience with the description of C. fragile has led us to revise the recommended criteria for an introduction of a new Cryptosporidium species name. C. fragile is the first species described and named from an amphibian host. Its prevalence of 83% (15/18) in black-spined toads within the 3 months after importation calls for strict quarantine measures and import regulation for lower vertebrates.