Single Oocyst Research Articles

Pathology caused by three species of Eimeria that infect the turkey with a description of a scoring system for intestinal lesions

ABSTRACT Three-week-old turkey poults were infected with pure lines of three species of Eimeria (E. adenoeides, E. gallopavonis, and E. meleagrimitis) recently isolated from commercial turkey farms. The lines had been propagated from a single oocyst and identified by species-specific PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Five to six days after infection their intestines were removed and examined for the presence of intestinal lesions. A description and review of the pathology caused by these parasites is provided, and a scoring system developed by which the severity of the lesions can be evaluated. The system is similar to that described by Johnson, J. and Reid, W. M. [1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology, 28, 30–36] for chickens in which a score of zero to four is assigned to lesions of increasing severity. The intestinal lesions observed here, and their assigned scores, are supported by representative illustrations. It is hoped that they may prove a useful tool for evaluating the pathology caused by E. adenoeides, E. gallopavonis, and E. meleagrimitis in the turkey. RESEARCH HIGHLIGHTS A scoring system has been developed for intestinal lesions caused by three species of Eimeria that infect the turkey. The lesions attributable to these species are illustrated.

Isolation, genotyping and subtyping of single Cryptosporidium oocysts from calves with special reference to zoonotic significance

The ability of the small-subunit ribosomal RNA (SSU rRNA) based nested PCR and Restriction Fragment Length Polymorphism (PCR-RFLP) to identify and genotype a single Cryptosporidium oocyst isolated from bovine faecal samples was evaluated in this study. In addition, subtyping was carried out by sequencing the 60 kDa glycoprotein (gp60) gene from the same single oocyst. Faecal samples were collected from 40 pre-weaned calves (5–20 days old) from 7 dairy farms located in 3 different counties within the Finger Lakes region of Upstate New York. All the samples were microscopically positive for Cryptosporidium spp. A total of 400 Cryptosporidium oocysts (10 single oocysts from each calf sample) were individually isolated and analyzed using a nested PCR targeting the SSU rRNA gene. The SSU rRNA gene was amplified in 324 (81%) individual oocysts. All SSU rRNA amplified individual oocysts DNA was genotyped using PCR-RFLP. C. parvum was the only identified species; 107 single oocysts generated PCR products from the A gene, 18 generated PCR products from the B gene and 199 generated PCR products from both. Sequence analysis of the gp60 gene in 99 individual oocysts revealed the presence of only subtype IIaA15G2R1 with 99.4–100% and 99.1–100% identity of nucleotides and amino acids, respectively. These sequences were identical (100%) in oocysts from 35 calves and exhibited mutations in the non-repeat region of the gp60 gene in those of 5 other calves. The examination of DNA from individual oocysts with genotyping and subtyping tools provides methodology to more clearly define the genetic characteristics of Cryptosporidium spp. on farms and within individual animals.

Prevalence of Eimeria Species in Camels (Camelus dromedarius) from Egypt and Variability in Structure of Eimeria cameli Oocysts

Coccidiosis is a common disease of camels, and camels are important for the economy of Asia and the Arabian Peninsula. Little is known regarding the prevalence of coccidian parasites in camels in Egypt. Fecal samples collected from the rectums of 200 camels at the Cairo slaughterhouse were processed using the sucrose flotation technique. Eimeria species oocysts were found in 38%. Three Eimeria species were identified: Eimeria cameli-like in 31%, Eimeria rajasthani in 18%, and Eimeria dromedarii in 14%. The morphology of E. rajasthani and E. dromedarii oocysts was identical to that in literature. However, there was great variability in size and structure among E. cameli oocysts; oocysts were 70-100 μm long and truncate to ovoid. Four morphotypes (types 1 to 4) were recognized. Types 1 and 2 oocysts had similar truncate ovoid shape and were dark brown, but their shape indices were different. Both types could be easily distinguished from type 3 (elongate ovoid and light brown). All oocysts were enclosed in a transparent outer covering (capsule), although this capsule was barely seen in types 3 and 4. An extension from the capsule situated in front of the micropyle, referred to as polar cap-like structure (PCL), was characteristic for types 1 and 2. The PCL of type 1 resembled the crown, while in type 2 it looked like a small thickening with a smooth top. The PCL was absent in types 3 and 4 oocysts. The latter was found only in a single oocyst. Experimental infections with E. cameli oocysts and molecular studies are needed to determine whether the monotypes described here are different species or strain variations or both.

Cecal coccidiosis in turkeys: Comparative biology of Eimeria species in the lower intestinal tract of turkeys using genetically typed, single oocyst-derived lines.

Differentiating the Eimeria species causing cecal coccidiosis in turkeys is challenging. To obtain benchmark biological data for Eimeria gallopavonis Hawkins 1952 and Eimeria meleagridis Tyzzer 1929 and to support the stability of the species concept for each, genetically typed, single oocyst-derived lines of E. gallopavonis Weybridge strain and E. meleagridis USAR97-01 were used to redescribe the biological, pathological, and morphological features of these parasites. Oocysts of E. meleagridis and E. gallopavonis overlap in dimensions, but oocysts of the former have a single polar granule compared with multiple in the latter. Mature first-generation meronts of E. gallopavonis were observed histologically as early as 48h post-inoculation alongside the villi in jejunum (before and after Meckel's diverticulum), ileum, cecal neck and rectum, but not cecal pouches. Three asexual cycles were observed suggesting that early workers apparently overlooked one asexual cycle. Examination of endogenous development of a culture labeled "Eimeria adenoeides Weybridge strain" suggested that this strain (found in a number of publications as a large oocyst strain of "Eimeria adenoeides") matched the species description of E. gallopavonis and so has been renamed herein. Macroscopic lesions induced by E. gallopavonis consisted of caseous material distally from posterior of the yolk stalk through the remaining intestinal tract, excluding the cecal pouches. For E. meleagridis, only the first asexual generation was observed outside of the cecal pouches within the jejunum around the yolk stalk. Second- and 3rd-generation asexual stages developed almost exclusively in the cecal pouches (but not cecal necks). Macroscopic lesions described for E. meleagridis were similar to those of E. adenoeides. Marked corrugation of the cecal serosal surface was observed. Cecal pouches contained creamy colored, caseous material varying from loose material to granular. Distinguishing features of the Eimeria species infecting the lower part of the small intestine are summarized in the present study, and new type specimens were designated for E. gallopavonis and E. meleagridis to provide a stable reference for future work with these parasites.

Detection and molecular status of Isospora sp. from the domestic pigeon (Columba livia domestica)

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0–27.2)×24.7 (23.4–26.0) μm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5–20.5) and 11.2 (10.2–12.1) μm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia.

Ophryocystis anatoliensis sp. nov., a new neogregarine pathogen of the chrysomelid beetle Chrysomela populi

Chrysomela populi (Coleoptera: Chrysomelidae) is the most abundant and most important pest species that causes damage to poplar trees. Members of the family Chrysomelidae are frequently infected by protist pathogens but no neogregarine has been reported to date at the species level. In the present study we identify a new neogregarine pathogen from the chrysomelid C. populi. The infection was observed in the Malpighian tubules of adult beetles. A reddening of the Malpighian tubules was the most distinctive symptom of the infection. Single fusiform oocysts (9.8×4.7μm) were formed within a gamontocyst. The polar plugs were very thin, varying from 380 to 525nm in thickness. The oocyst wall was smooth and also quite thin (90–120nm). Morphological and ultrastructural characteristics of the pathogen indicate that the described neogregarine in C. populi is clearly different from known Ophryocystis species which infect coleopterans. Therefore, the neogregarine pathogen was determined to be a newly discovered species and named Ophryocystis anatoliensis sp. nov.

Evaluation of the Doodlebug: A Biochip for Detecting Waterborne Pathogens

Doodlebug is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label- and reagent-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. Because imaging Raman microscopes offer spatial resolution as fine as 250 nm, spectra can be collected from individual microorganisms captured by the pixels. This exquisite sensitivity eliminates the need for labels such as fluors or enzymes, or reagents such as those used in PCR; and, thereby, eliminates the potential for false responses from environmental constituents that either mimic or mask the signals from labels or reagents. The WERF program showed that SERS, like other whole-organism fingerprinting techniques, is very specific. Studies with representative strains of six Legionella species, recently-passaged oocysts from six C. parvum genotype 2 strains, three genotype 1 strains, and a C. meleagridis strain, plus a Giardia sample, indicated that SERS could identify the bacteria and oocysts at the species and subspecies levels. Therefore, Doodlebug holds promise for individually identifying pathogens, even when organisms that cross-react with the capture biomolecules are present in a sample. The fingerprints that were used in oocyst identification were routinely collected from a single oocyst within 60 seconds. Experiments involving the impact of environmental and water treatment conditions suggest that the Doodlebug approach will be able to differentiate between viable and nonviable, and possibly injured, organisms. It appears that the SERS fingerprint may even be useful in determining the ‘age’ of an oocyst; e.g., whether it is too old to be infectious. The WERF program culminated in a demonstration of the simultaneous detection of two very different microorganisms (i.e., C. parvum oocysts and Bacillus spores) present in a mixture. Although oocyst capture was selective, some spores adhered nonspecifically to the anti-oocyst MAb surface. This showcased Doodlebug's ability to eliminate false responses due to nonspecific adherence or cross-reactivity, since the spores were readily differentiated from the oocysts on the basis of their SERS fingerprints. This title belongs to WERF Research Report Series ISBN: 9781843396680 (Print) ISBN: 9781780403175 (eBook)

Profil Saraf Nitrergik Sekum Ayam Pedaging yang Diinfeksi Eimeria tenella

Neurotransmitter found in the enteric nervous system that plays an important role in a variety ofenteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to bedependent on the manner by which inflammation is caused. This study was performed to determine theprofile of neural nitrergic with Nicotinamide Adenine Dinucleotide Phosphate-diaphorase (NADPH-d)staining in the cecum of broiler infected by Eimeria tenella (E. tenella). Thirty six health free of coccidiosisbroiler one-day old chickens (DOC) used in this study. All of broiler adapted for 7 days with food and drinkgiven ad libitum then divided into three groups, each group consisted of 12 broilers. The 1st group wascontrol only given orally 1.0 ml of distilled water/ heads , while 2nd group was infected with a single dose of5x103 oocysts/ head orally, and 3rd group was infected with a single dose of 2x104 oocysts/ head orally. Onday 7 post infection, all of chickens were fasted for 12 hours then were euthanized and cecum was taken.Lesion score of cecum was assessed. Furthermore, tissue of the coecum was prepared for hisochemicalusing NADPH-d staining to determine morphology, and morhometric of nitrergic neurons. The resultshown that cecum lesion score of group I is 0, group II is +2, group III is +3. Neuron density in the cecum ofgroup I is 2794.96+4.65 neuron/cm2, group II is 4930.36+63.73 neuron/cm2 and group III is 7892.31+44.97 neuron/cm2. Neuron density of group II and III increased signifcantly (p<001) than group I. Nitrergic neurondesity of group III increased significantly (p<0.05) than that of group II. It was concluded that the infectionof E.tenella led to increase the number of neutrl mienteric nitrergic of the cecum. The higher lesion score ofcecum led to increase the number of mienteric nitrergic neuron.

Complete sporogony of Plasmodium relictum (lineage pGRW4) in mosquitoes Culex pipiens pipiens, with implications on avian malaria epidemiology.

Plasmodium relictum (lineage pGRW4) causes malaria in birds and is actively transmitted in countries with warm climates and also temperate regions of the New World. In Europe, the lineage pGRW4 has been frequently reported in many species of Afrotropical migrants after their arrival from wintering grounds, but is rare in European resident birds. Obstacles for transmission of this parasite in Europe have not been identified. Culex quinquefasciatus is an effective vector of pGRW4 malaria, but this mosquito is absent from temperate regions of Eurasia. It remains unclear if the lineage pGRW4 completes sporogony in European species of mosquitoes. Here we compare the sporogonic development of P. relictum (pGRW4) in experimentally infected mosquitoes Culex pipiens pipiens form molestus, C. quinquefasciatus, and Ochlerotatus cantans. The pGRW4 parasite was isolated from a garden warbler Sylvia borin, multiplied, and used to infect laboratory-reared Culex spp. and wild-caught Ochlerotatus mosquitoes by allowing them to take blood meals on infected birds. The exposed females were maintained at a mean laboratory temperature of 19 °C, which ranged between 14 °C at night and 24 °C during daytime. They were dissected on intervals to study the development of sporogonic stages. Only ookinetes developed in O. cantans; sporogonic development was abortive. The parasite completed sporogony in both Culex species, with similar patterns of development, and sporozoites were reported in the salivary glands 16 days after infection. The presence of sporogonic stages of the lineage pGRW4 in mosquitoes was confirmed by PCR-based testing of (1) the sporozoites present in salivary glands and (2) the single oocysts, which were obtained by laser microdissection from infected mosquito midguts. This study shows that P. relictum (pGRW4) completes sporogony in C. p. pipiens at relatively low temperatures. We conclude that there are no restrictions for spreading this bird infection in Europe from the point of view of vector availability and temperature necessary for sporogony. Other factors should be considered and were discussed for the explanation of rare reports of this malaria parasite in Europe.

Eimeria collieie n. sp. (Apicomplexa:Eimeriidae) from the western long-necked turtle (Chelodina colliei)

A new species, Eimeria collieie n. sp., is described from the western long-necked turtle (Chelodina colliei). Sporulated oocysts (n = 35) are spherical to subspherical, with colourless single layer oocyst wall, 0.6 ± 0.2 (0.4–0.7) µm thick. Oocyst with elongated ellipsoid sporocysts. Oocyst length, 29.8 ± 0.4 (28.2–31.0) µm; oocyst width, 29.4 ± 0.3 (28.0–30.8) µm; oocyst length/width (L/W) ratio, 1.0 ± 0.03 (1.0–1.05). Micropyle, oocyst residuum and polar granule were absent. Sporocysts with sporocyst residuum and 2 sporozoites. Sporocyst length, 21.6 ± 0.4 (21.2–22.0) µm; sporocyst width, 6.0 ± 0.3 (5.7–6.3) µm; sporocyst L/W ratio, 3.6 ± 0.2 (3.4–3.8). Stieda, parastieda and substieda bodies were absent. Sporozoite length, 14.0 ± 0.2 (13.8–14.2) µm; sporozoite width, 2.6 ± 0.2 (2.4–2.8) µm; sporozoite L/W ratio, 5.46 ± 0.10 (5.4–5.6). Molecular analysis was conducted at three loci: the 18S and 28S ribosomal RNA (rRNA), and the mitochondrial cytochrome oxidase gene (COI). At the 18S rRNA locus, E. collieie n. sp. shared 96.4% and 98.3% genetic similarity to E. ranae (GenBank accession number: EU717219) and E. arnyi (AY613853) respectively. At the 28S rRNA locus, E. collieie n. sp. shared 91.6% genetic similarity to E. papillata (GenBank accession number: GU593706) and phylogenetic analysis at this locus placed E. collieie n. sp. in aseparateclade. At the COI locus, E. collieie n. sp. shared 92.7% genetic similarity to Eimeria setonicis (GenBankaccession number: KF225638) from a quokka (Setonix brachyurus) in Western Australia. Reptile-derived sequences were not available for the 28S rRNA and the COI loci. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that, to date, has only been found in western long-necked turtles.

A new species of Isospora (Apicomplexa: Eimeriidae) from eastern coachwhip, Coluber flagellum flagellum (Reptilia: Ophidia) from Oklahoma.

Between June 2013 and August 2014, four eastern coachwhips, Coluber flagellum flagellum were collected from Arkansas (n = 2) and Oklahoma (n = 2) and their faeces examined for coccidian parasites. One (25%) harboured an isosporan that we describe here as new. Oocysts of Isospora kiamichiensis sp. n. were spheroidal to subspheroidal with a uni-layered wall and measured (length × width, L × W) 25.0 × 22.2 μm, with an L/W ratio of 1.1. A micropyle, oocyst residuum and polar granule were absent. Sporocysts were ovoidal, 13.9 × 9.4 μm, with an L/W ratio of 1.5. A pronounced, button-like Stieda body was present as well as a substieda body. A sporocyst residuum was present as dispersed granules. This is the first isosporan and fourth coccidian reported from eastern coachwhip snakes. In addition, a single oocyst of an unknown choleoeimerian was recovered from this host.

Biological characteristics of Chinese precocious strain of eimeria acervulina and its immune efficacy against different field strains.

In this study, the biologic characteristics of one experimental precocious strain of Eimeria acervulina and seven field isolates from different geographic locations in China were compared, and the immune efficacy of two precocious strains against coccidiosis in chickens was assessed to explore their potential use as coccidiosis vaccines. All the different strains were purified by single oocyst separation and their monospecificity was confirmed using E acervulina-specific PCR assays. The average sizes of E. acervulina oocysts were 18.28-20.19 X 14.09-14.79 microm and the shape indexes were from 1.28 to 1.40. The prepatent periods ranged from 93 to 115 hr, except for the Heyuan precocious strain (HYP; 75 hr). Chickens infected with Huadu field strain (GHD) produced the highest oocyst output whereas HYP induced the lowest level. When inoculated with 50,000 sporulated oocysts or more, the average weight gains of infected chickens were reduced, with apparent clinical symptoms. To assess the immunogenicity of precocious strains HYP and Baoding (BDP), birds were orally immunized and challenged with seven different field strains of E. acervulina. Body weight gain, fecal oocyst output, and gut lesion scores were compared to evaluate their vaccine potential. The results showed that the average body weight gains of chickens in all the vaccinated and challenged groups were higher than those of nonvaccinated and challenged groups. In general, oocyst shedding was reduced 34.39%-95.31% and gut lesion scores decreased 31.03%-86.21% compared with unvaccinated and challenged control chickens. In summary, this study indicated that the precocious strains of E. acervulina could induce a protective immune effect with various responses against coccidiosis caused by different field strains.

Re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria adenoeides Moore and Brown, 1951.

The Guelph strain of Eimeria adenoeides was obtained from a commercial turkey flock in Ontario, Canada, in 1985. Single oocyst derived lines of E. adenoeides were propagated, and one of them used to re-describe biological and morphological features of E. adenoeides in the turkey. Oocysts of this strain are within the lower size ranges in the original species description reported by Moore and Brown (1951); oocysts of the Guelph strain averaged 18.7 ± 1.4μm (16.7-22.5) by 14.3 ± 0.9μm (13-16.2, n = 30) with a shape index (SI) of 1.3 ± 0.1. It is possible that the original species description was based, at least in part, on a mixed culture of two or more Eimeria species. Immature first-generation meronts of E. adenoeides Guelph strain were observed histologically at 32h post-infection in the ileum and cecal neck. Early studies reported only two asexual generations suggested that first asexual cycle observed at 32h post-infection was overlooked. In the present study, three asexual generations were observed before the start of gametogony. The Guelph strain is also characterized by a prepatent period of 112h. The Guelph strain of E. adenoeides is a highly pathogenic coccidium that forms classic cecal lesions, including prominent caseous cecal cores, during moderate to severe infections. The maximum output of oocysts (1.77 × 10(7) per bird) was obtained from birds inoculated with 1 × 10(3) oocysts; maximum fecundity (1.55 × 10(5) oocyst shed per oocyst inoculated) was obtained with an inoculation of 1 × 10(2) oocysts, but fecundity dropped dramatically as the inoculation dose increased. To promote stability of the E. adenoeides species concept, neotype specimens (a parahapantotype slides series and phototype) have been designated and deposited for future reference.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists.

Endoparasites of the European beaver (Castor fiber L. 1758) in north-eastern Poland

Abstract Parasitological examination after necropsies of 48 European beavers from Podlaskie and Warmisko-Mazurskie provinces were performed between April 2011 and November 2012. All helminthes were isolated from the contents of the gastro-intestinal tract and their species were determined. In addition, blood samples and faeces were examined. All beavers were infected with six species of parasites. Stichorchis subtriqetrus trematodes were found in 93.7% of animals. They were localized mainly in the caecum, less in the colon, and single juvenile parasites were found in the small intestine. The intensity of infection ranged from two to 893 parasites. Travassosius rufus nematodes (10-4336 specimens) were present in the stomach of 68.7% of the beavers. In the small intestine of four (8.3%) beavers, two-six specimens of Psilotrema castoris were found. This is the first record of this species in Poland and the third of its discovery in the world. Furthermore, in the small intestine of one beaver, two Trichostrongylus capricola nematodes were detected. In the liver of one beaver, pathological changes caused by hydatid cestode Echinococus granulosus occurred. Inflammatory changes of the gastric mucosa caused by Travassosius rufus and of caecum caused by Stichorchis subtriquertus, were observed. Coproscopy was performed with the use of Baermann, flotation, and decantation methods. All results of Baermann method were negative. Examinations with flotation and decantation methods confirmed necropsy findings. Using the flotation method, single oocysts of Eimeria sprehni in one beaver were detected. A blood test conducted by Kingston and Morton method did not reveal the presence of protozoa or microfilariae.

Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis

Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata.

Biological re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria meleagrimitis Tyzzer 1929

For the purpose of re-describing the Eimeria species that infect the turkey (Meleagris gallopavo) and to establish benchmark biological information linked to genetic markers for each species, a strain of Eimeria meleagrimitis Tyzzer 1929 was obtained from a litter sample from a turkey farm in Minnesota, USA in 2008. Multiple pure lines were derived by infecting turkey poults with a single oocyst; one of these lines was then used to re-describe biological and morphological features of E. meleagrimitis in the turkey and to designate a neotype of E. meleagrimitis in the turkey. Oocyst morphometrics of this line matched those of this species as originally described by Tyzzer (Am J Hyg 10:269-383, 1929). Three asexual generations of merogony (the first generation of meronts large in size and the second and third generations small) were detected in the intestines before the onset of gametogony; no developmental stages were detected in the cecal pouches. No mortality was induced by this line of E. meleagrimitis even when turkey poults were infected with high doses of oocysts (up to 5 × 10(5) oocysts/bird) and despite the ability of E. meleagrimitis to induce severe mucosal damage in the upper and middle duodenum. Macroscopic lesions were characterized to provide a graded lesion scoring guide that should assist assessment of the severity of infections with this species in infected turkeys. The pathogenicity of the strain was investigated, and a significant reduction in weight gain and feed conversion ratio was observed with doses of 10(4) oocysts/bird or more. The maximum yield of oocysts in the feces was obtained when birds were inoculated with 5 × 10(3) oocysts.

English

Toxoplasmosis is a widely distributed protozoan disease caused by Toxoplasma gondii. The infective stages of T. gondii are capable of infecting a variety of vertebrates including humans. Domestic and wild felids are capable of serving as definitive hosts and T. gondiioocysts are excreted in their faeces. Toxoplasmosis is caused by ingesting T. gondiioocysts from contaminated water or foodstuff or by consuming T. gondii tissue cysts from infected hosts. However, the impact of oocysts on toxoplasmosis epidemiology needs to be specified because they are suspected to be associated with T. gondii seroprevalence in some emerging outbreaks of acute toxoplasmosis in humans from soil or water. They are probably responsible for a significant part of infections in animals that could be later consumed by humans. Detection of T. gondii oocysts in environmental samples is great challenge as this coccidian parasite can be responsible for severe infections in humans and animals via ingestion of a single oocyst from contaminated water, soil, fruits or vegetables. The present study was designed to develop methods for the detection of oocysts from water, soil and food. The highest prevalence was reported from water around farms (13%) followed by lakes and pools (9%) and water reservoirs (7%). The lowest rates resulted from drinking water and tubewells (6% each). Overall prevalence ofT. gondii contamination was higher in vegetables (5.6 %) than fruits (4 %). The highest prevalence of T. gondii contamination was noted in gardens and backyards of homes and gardens, that is, 20 and 20%, respectively. In these places, cats often defecate and become a source of infection. Then followed by public enclosures where infection was 14.3%. The lowest (13.3%) contamination was noted in homes of urban areas. T. gondiiinfection in rural areas was the highest (20%) in home back yards followed by homes (16.7%) then public enclosure (14.5%) whereas the lowest (13.3%) at gardens of rural areas. Key words: Toxoplasmosis, Toxoplasma gondii, detection, water, foodstuff.

Toxoplasma gondii sexual cross in a single naturally infected feline host: Generation of highly mouse-virulent and avirulent clones, genotypically different from clonal types I, II and III

Tachyzoite clones obtained from a single Toxoplasma gondii oocyst field sample were genotyped and characterized regarding mouse virulence. PCR-RFLP genotyping of tachyzoites initially isolated from interferon-γ-knockout (GKO) mice, BALB/c mice and VERO cell culture using the nine independent, unlinked genetic markers nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico revealed mixed T. gondii infections showing combinations of type II and type III alleles at different loci. Forty-five individual clones were obtained from all mixed T. gondii tachyzoite cell cultures by limiting dilution. Sixteen T. gondii clones showed type III alleles at all loci and 29 clones displayed a combination of type II and type III alleles at different loci. Five clone groups were identified in total, four of which include T. gondii clones that showed a non-canonical allele pattern and have never been described in natural infections before. All tested clones, except two, were highly virulent in BALB/c mice. The isolation of different non-canonical T. gondii clones originating from an oocyst sample of a single naturally infected cat demonstrate that sexual recombination as well as re-assortment of chromosomes via a sexual cross of T. gondii occur under natural conditions and result in the emergence of clones with increased virulence in mice.

Accurate analysis of prevalence of coccidiosis in individually identified wild cranes in inhabiting and migrating populations in Japan

Eimeria gruis and E. reichenowi cause coccidiosis, a major parasitic disease of cranes. By non-invasive molecular approaches, we investigated the prevalence and genetic characterization of pathogens in two Japanese crane habitats; one is Hokkaido inhabited by the endangered red-crowned crane, and the other is Izumi in Kyushu where populations that consist mainly of vulnerable hooded and white-naped cranes migrate in winter. The non-invasively collected faecal samples from each wintering population were first subjected to host genomic DNA-targeted analyses to determine the sample origin and avoid sample redundancy. Extremely high prevalence was observed in the Izumi populations (> 90%) compared with the Hokkaido population (18-30%) by examining 470 specimens by microscopy and PCR-based capillary electrophoresis (PCR-CE), using genetic markers in the second internal transcribed spacer (ITS2). Correspondence analysis of PCR-CE data revealed differences in community composition of coccidia between hooded and white-naped cranes. 18S rRNA and ITS2 sequences were determined from single oocysts excreted by red-crowned and hooded cranes. Phylogenetic analysis of 18S rRNA suggested that E. reichenowi was polyphyletic while E. gruis was monophyletic. Together with PCR-CE data, these results indicate different host specificity among the E. reichenowi type. Our data suggest that E. reichenowi comprises multiple species.