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.

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.

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.

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.

Biology and pathogenicity of Eimeria spinosa Henry, 1931 in experimentally infected pigs.

A single-species isolate of E. spinosa from a diarrheic weaned pig was used to determine the endogenous development and pathogenicity of this swine coccidium. Seven out of 14 inoculated pigs developed endogenous stages or passed oocysts of E. spinosa in their feces. Immunosuppressive treatment with cyclophosphamide had no effect on the susceptibility to infection with E. spinosa in young pigs. The endogenous stages developed within the apical cytoplasm of the enterocytes lining the distal part of the villi in the posterior jejunum. The asexual development comprised three generations of meronts, which were seen at 5, 7 and 9 days post-infection (DPI). Meronts of the first generation measured 6-8 microns and produced 10-14 merozoites 4-6 microns in length. The second generation of meronts measured 6-8 microns and contained 10-20 merozoites 4-6 microns in length. Third generation mature meronts (8-10 microns) on DPI 9 contained 12-20 merozoites measuring 5-7 microns, which were more crescent-shaped and less blunt than the merozoites at 5 and 7 DPI. Merogony continued after formation of the gametes and the first fully developed macrogametes (10-14 microns), microgametes (9-12 microns), and oocysts were also seen at 9 DPI. The prepatent period was 8 or 9 days, but the patent period was not determined. In the present study E. spinosa infection did not produce overt clinical signs. Pathological changes consisted of an inflammatory infiltration in the lamina propria of the posterior jejunum, Peyer's patches activation and sporadic erosions scattered at the villous tips. No villous atrophy in association with a large number of endogenous stages was observed.

Pathogenicity of experimental caryosporosis.

Pathogenicity of the coccidia C. bigenetica and C. simplex was studied in experimentally inoculated pigs, goat kids (untreated and immunosuppressed) and severe combined immunodeficient (SCID) mice. The major pathological changes of caryosporosis were similar in all inoculated animals. In pigs and goat kids, caryosporosis was self-limiting, with clinical responses that included focal swelling and erythema of the muzzle, snout, jaws, cheeks, eyelids, bases of the ears, backs of the necks, scrotum, external genitalia of females, legs and footpads. Histopathological changes were characterized by involvement of the cutaneous mononuclear phagocyte system with an inflammatory exudate containing numerous macrophages, especially around the root sheaths, sensory nervous corpuscles of the hair follicles and surrounding dermal free nerve endings. The tactile hair follicles in the muzzle, snout and upper jaw were most severely changed. In SCID mice, inoculation with C. bigenetica or C. simplex caused a severe, fatal, systemic disease characterized by dissemination of numerous caryosporan developmental stages into the host mononuclear phagocyte system. This study presents evidence that both caryosporan species tested caused similar clinical signs and lesions of dermal coccidiosis in the mammalian secondary hosts.

Multiple origin of the dihomoxenous life cycle in sarcosporidia.

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

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.

Pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets.

The pathogenesis of intestinal cryptosporidiosis was studied in 52 conventionally reared and 20 gnotobiotically reared piglets by inoculation with different doses of Cryptosporidium parvum oocysts. The prepatent period of C. parvum in both groups of animals were variable, depending on the number of oocysts administered. The patent period of C. parvum in conventionally reared piglets was 8 or 9 days; in gnotobiotic piglets cryptosporidia were found in feces until Day post infection (DPI) 16, when the last piglet was necropsied. Cryptosporidiosis in conventionally reared piglets is a self-limited diarrheal disease associated with morphological changes within the intestine. The most severe lesion was seen in the posterior jejunum and ileum from DPI 3 to DPI 7, and consisted of villous atrophy, crypt hyperplasia and inflammatory infiltration in the lamina propria. In gnotobiotic piglets cryptosporidia induced severe enterocolitis which occurred at least until DPI 16. The characteristics of enteric lesions were similar to those found in conventionally reared piglets. Intestinal cryptosporidiosis in both groups of animals shifted in the course of infection in the caudal direction and terminated in the large intestine. Examination by scanning electron microscope showed that infected absorptive cells had thicker and longer microvilli than those on non-infected cells; neighboring non-infected cells were hypertrophic, bulbously protuberant with minute microvilli with no distinct intercellular borders. Numerous cryptosporidia in the heterotopic glandular epithelium in the submucosa of cecum and colon on DPI 9 and 10 were found. No differences in the location and degree of cryptosporidial infection between colostrum-fed and colostrum-deprived conventionally reared piglets were found. Sow's colostrum does not appear to protect piglets from C. parvum infection. The role of intestinal microflora in the pathogenesis of cryptosporidiosis in piglets is discussed.

Experimental cryptosporidiosis in kids.

The clinical, pathological and parasitological features of cryptosporidiosis resulting from experimental inoculation with 6 x 10(6) Cryptosporidium parvum oocysts were studied in kids. Decreased appetite and depression became apparent 72 h post inoculation. Subsequently watery feces with clumps of mucus and color changes from brown to yellow were observed. The mean duration of diarrhea was 4.2 days. Oocyst shedding started 4 days post inoculation (DPI), started to decrease at 7 DPI, and lasted until 12 DPI. The evidence of high infectivity and fast transmission of C. parvum oocysts was observed under standard zoohygienic conditions. The characteristics of intestinal lesions were similar to those found in other neonatal ruminants infected with C. parvum. The most severe lesions were seen in the posterior jejunum and ileum from 3 to 7 DPI, characterized by villus atrophy, villus blunting, fusion of atrophic villi, crypt hyperplasia, inflammatory infiltration in the lamina propria, and metaplasia of mucosal epithelium. Scanning electron microscopy of ileal epithelium revealed ultrastructural changes on the surface of intestinal mucosa. No cryptosporidia or associated pathological lesions were found in the large intestine or other tissues. The distribution of cryptosporidia in the intestine and number of cryptosporidia per ileal villus on different DPI were also estimated for detailed characterization of the infection in kids as a model for experimental cryptosporidiosis.

Coccidiosis in goats in the Czech Republic.

An observational study was conducted to determine coccidial infections in goats of 13 farms in the Czech Republic. The prevalence of oocysts of Eimeria species in kids (less than 3 months old), weaned but not served goats (from 3 months to 1 year), and adult goats (1 year or more) was determined. Nine Eimeria species were identified in fecal samples by Sheather's sugar flotation technique. The overall prevalence of Eimeria oocysts in fecal specimens was 92.2%. Eimeria arloingi was the most common species with an overall prevalence of 84%, followed by E. hirci (63%) and E. ninakohlyakimovae (56%). Other species present were E. christenseni (55%), E. alijevi (36%), E. caprina (25%), E. aspheronica (12%), E. capriovina (6%) and E. jolchijevi (2%). Two or more Eimeria species were detected in 88% of the samples. The most prevalent species in kids was E. arloingi, while in weaned but not served and adult goats E. ninakohlyakimovae was the most frequently found. The number of oocysts excreted was generally lower in adult goats (2567.3+/-12678 OPG), whereas higher number oocyst per gram of feces (OPG) were found in kids (18565+/-24888 OPG). Clinical coccidiosis was detected in two farms, and E. arloingi and E. ninakohlyakimovae were implicated as its cause. Disease was observed in kids 2 to 4 weeks after weaning and watery feces with clumps of mucus, and color changes from brown to yellow or dark tarry, weight loss, and dehydration were the most conspicuous clinical signs. At necropsy, macroscopic changes included mucosal hemorrhages and whitish nodular polyps in the jejunum were found. Histopathological changes were characterized by local hypertrophy and hyperplasia of intestinal villi, villus blunting and inflammatory infiltration in the lamina propria. Numerous developmental stages of the parasites were observed in enterocytes and lacteals of intestinal villi.

Experimental giardiasis in goat kids.

The clinical, pathological and parasitological features of giardiasis resulting from experimental inoculation with 3 x 10(6) Giardia cysts were studied in goat kids. All experimentally inoculated goat kids given Giardia cysts became infected. Three of the eight inoculated kids had decreased appetite, formless feces and become slightly depressed beginning 7 or 8 days post inoculation. The mean duration of the appearance of abnormal feces was 6 days. Irregular and intermitted cysts shedding started after prepatent periods of 6-10 days and lasted throughout this study (10 weeks). The evidence of high infectivity and fast transmission of Giardia were observed under standard zoohygienic conditions. The characteristics of intestinal lesions were similar to those found in other hosts infected with Giardia. The most severe lesions were seen in the duodenum and proximal jejunum, and consisted of moderate villus atrophy, villus blunting, crypt hyperplasia and inflammatory infiltration in the lamina propria. Scanning electron microscopy revealed ultrastructural alterations in the microvillus border of enterocytes. Mucosal smears and histological sections of the gall bladder displayed Giardia trophozoites and gall bladder epithelium hyperplasia together with bile ductular proliferation in the liver tissue in two kids.

Role of acquired immunity and natural age resistance on course of Isospora suis coccidiosis in nursing piglets.

Thirty-two piglets from three litters were experimentally inoculated with 200000 sporulated oocysts of Isospora suis at 3 days of age and/or rechallenged at 19 days of age or primary inoculated at 19 days of age, to compare the role of acquired immunity and natural age resistance on the course of coccidiosis. Twelve piglets were not inoculated and served as a control. Following challenge, the signs of coccidiosis characterised by clinical symptoms, oocysts shedding and weekly weights were similar to those which occurred in piglets primary inoculated at 19 days of age. This comparison suggests that maturation of non-specific components of the immune system plays a more important role in the resistance of neonatal piglets to I. suis infection than specific immune mechanisms.

Concurrent infection of enterocytes with Eimeria scabra and other enteropathogens in swine.

Bacteria were detected in the enterocytes of the distal jejunum in weaned pigs on Days 7 and 9 post-infection (DPI) with Eimeria scabra in addition to the developmental stages of the coccidia. Short rod-shaped bacteria were identified in approximately 60% of the enterocytes that contained developmental stages of E. scabra. No such bacteria were observed in cells where coccidia were absent. Gamonts of cryptosporidia were also observed within the microvillous zone of the enterocytes in the distal jejunum of weaned pigs on DPI 9 with E. scabra. Cryptosporidia were present only in enterocytes harbouring stages of E. scabra. Chlamydial particles were also found in the cytoplasm of enterocytes 7 DPI with E. scabra. The presence of other enteropathogens exclusively in the enterocytes containing developmental stages of coccidia suggests that the coccidium E. scabra facilitates the invasion and development of bacteria, cryptosporidia and chlamydia in the enterocytes.

Infectivity of Cryptosporidium muris isolated from cattle.

The infectivity of a bovine isolate of Cryptosporidium muris for various animals was studied by transmission experiments. Neonatal BALB/c mice, adult BALB/c mice, SCID mice, common voles (Microtus arvalis), bank voles (Clethrionomys glareolus), common field mice (Apodemus sylvaticus), Mongolian gerbils (Meriones unguiculatus), desert gerbils (Gerbilus gerbilus), guinea pigs, rats, rabbits and goats were used to test the infectivity of this isolate. Among these host species, only Mongolian gerbils were susceptible to the infection and discharged C. muris oocysts in their faeces. The prepatent period for 8-week-old Mongolian gerbils was 15-19 days, the patent period varied between 18 and 36 days. More protracted chronic infections have been observed in gerbils immunosuppressed with methylprednisolone. No signs of clinical illness or macroscopic changes were seen in infected gerbils. Cryptosporidial developmental stages were detected in the stomach, histopathological changes were characterized by epithelial hyperplasia and mucosal hypertrophy without inflammatory exudate. In spite of the fact that C. muris was able to infect gerbils, we do not consider gerbils to be a true hosts for C. muris of cattle origin. Based on our results, we suggest that significant differences in host specificity of individual C. muris isolates exist, and that wild rodents are not reservoir for C. muris infection of cattle.

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.

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species.

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice

Sarcocystis muris possesses both diheteroxenous and dihomoxenous characters of life cycle.

The cystozoites of Sarcocystis muris were infective to other mice after peroral inoculation. They transformed into gamonts and after fertilization underwent sporulation with the production of infectious oocysts/sporocysts in the lamina propria of the small intestine. The present study demonstrated that S. muris possesses both diheteroxenous and dihomoxenous life cycle and can be transmitted by the cannibalism among mice.