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Faecal samples from 514 kangaroos and wallabies representing 12 species of the genus Macropus were examined for oocysts of Eimeria spp. Six species of Eimeria were redescribed from their type hosts, and on the basis of finding homologous oocysts in the faeces of other Macropus spp., host ranges for these coccidia were extended. Eimeria hestermani Mykytowycz, 1964 is redescribed from M. giganteus (eastern grey kangaroo) and is described from M. fuliginosus (western grey kangaroo), M. rufogriseus (red-necked wallaby), M. dorsalis (black-striped wallaby), and M. eugenii (tammar wallaby). E. toganmainensis Mykytowycz, 1964 is redescribed from M. rufus (red kangaroo) and the host range is extended to M. giganteus, M. fuliginosus, M. rufogriseus and M. eugenii. E. wilcanniensis Mykytowycz, 1964 is redescribed from M. rufus, and the host range is extended to M. giganteus, M. fuliginosus and M. robustus (euro or wallaroo). E. macropodis Wenyon & Scott, 1925 is redescribed from M. rufogriseus, and is described from M. giganteus, M. fuliginosus, M. rufus, M. irma (western brush wallaby), M. parryi (whip-tailed wallaby), M. dorsalis, M. eugenii, and M. parma (parma wallaby). E. fausti Yakimoff & Matschoulsky, 1936, E. cunnamullensis Mykytowycz, 1964 and E. purchasei Mykytowycz, 1964 are synonymized with E. macropodis. E. marsupialium Yakimoff & Matschoulsky, 1936 is redescribed from M. giganteus, and from M. fuliginosus. E. gungahlinensis Mykytowycz, 1964 is redescribed from M. fuliginosus, and from M. giganteus. Seven new species of Eimeria are described. E. flindersi, new species, is described from M. eugenii, M. rufogriseus, and M. antilopinus (antilopine wallaroo). E. prionotemni, new species, is described from M. eugenii, M. parryi, M. rufogriseus, M. agilis (agile wallaby) and M. dorsalis. E. mykytowyczi, new species, is described from M. agilis, M. antilopinus, and M. parryi. E. parryi, new species, is described from M. parryi. E. yathongensis, new species, is described from M. fuliginosus and M. giganteus. E. parma, new species, is described from M. parma, and E. desmaresti, new species, is described from M. rufogriseus. E. kogoni Mykytowycz, 1964, and E. rufusi Prasad, 1960 are considered species inquirendae. The host-parasite associations of these coccidia, and of similar species of Eimeria in other genera of Macropodoid marsupials, are discussed in relation to the postulated phylogeny of the hosts.
Ova and free oxygen radical production and relative peripheral eosinophilia during single and concurrent infections in mice with P. berghei and N. brasiliensis were investigated. Prolonged helminth patent periods indicate that Nippostrongylus self-cure in concurrently infected mice was suppressed. Differential white blood cell determinations showed that the relative number of peripheral blood eosinophils steadily increased (P < or = 0.05) during a Nippostrongylus infection when compared to noninfected controls. Eosinophil levels in mice singly infected with P. berghei or concurrently infected with both parasites did not differ significantly from those of controls suggesting a suppression by Plasmodium of Nippostrongylus-induced eosinophilia. Generation of intestinal free oxygen radicals was indirectly assessed using the Thiobarbituric Acid Assay to measure malondialdehyde (MDA). Intestinal MDA levels were significantly elevated (P < or = 0.05) during single Plasmodium infections as well as concurrent infections while there was no change in MDA production during single Nippostrongylus infections. These results suggest suppression by Plasmodium of the immune response to Nippostrongylus, allowing prolonged patent periods. They also suggest that eosinophils play a role in self-cure while free oxygen radicals do not.
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Two monoclonal antibodies directed against a microneme antigen of Sarcocystis muris cyst merozoites (16/17 kDa band doublet) were used to isolate cDNA clones from a lambda ZAP expression library. Restriction analysis revealed that the inserts were highly similar, with sizes ranging between 1.8 and 2.3 kb. In addition, a full-length cDNA insert of 2.6 kb was obtained by hybridization screening. On Northern blots, a single mRNA species of 2.7 kb was detected by a cDNA-derived probe. Southern blot hybridization suggests that the gene is present as a single copy. The nucleotide sequence of the full-length clone contains a single reading frame with a coding capacity of 26.5 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide followed by a hydrophilic domain of unknown function, and the mature protein sequence. After purifying the 16/17 kDa antigen from cyst merozoites, a partial N-terminal amino acid sequence was obtained. Thus, the identity of the cDNA sequence was confirmed. The deduced sequence of the mature protein is predominantly hydrophilic and rich in cysteine (8.7%). Database searching suggested weak homologies of the hypothetical polypeptide to plasma kallikrein, tenascin and blood coagulation factors.
A genomic library of Sarcocystis cruzi sporozoite DNA was constructed in bacteriophage lambda gt10. Recombinant phages containing insert DNA were selected by growth on Escherichia coli strain C600 hflA150. Of 14 clones examined, 11 contained DNA inserts ranging in size from approximately 1.45 kilobase (kb) to 6.18 kb. Insert DNA from four of these clones specifically hybridized to 32P-labelled S. cruzi merozoite DNA. One of these insert DNA, clone SL41, was selected and labelled with 32P. This probe did not hybridize with the other ten DNA inserts nor with bovine cellular DNA, but it hybridized with sporozoite, merozoite and bradyzoite DNA preparations. The SL41 probe could detect merozoite DNA in as little as 17 ng total DNA. Genomic probes detecting developmental stages of Sarcocystis spp. could provide an improved means is diagnosis of acute bovine sarcocystosis.
Four crossbred pigs (Sus scrofa) were inoculated orally with Caryospora bigenetica oocysts derived from snake and mouse feces, and with C. bigenetica infected mouse tissue. One pig also was given i.m. injections of methylprednisolone acetate. All four pigs displayed clinical signs including erythema, edema, and lethargy. Caryocysts were observed histologically in numerous tissues including ear, tongue, jowl, shoulder, loin, intercostal, ham, hock, and feet. The four pigs each were butchered into six commercial cuts: shoulder, loin, side, ham, hock, and feet. Raw 10 g samples from each cut were bioassayed by pepsin digestion and s.c. inoculation into 12 Swiss-Webster mice (Mus musculus) and 12 cotton rats (Sigmodon hispidus). Seventeen of 24 mice and cotton rats exhibited clinical signs and C. bigenetica tissue infections. Remaining portions of the six commercial cuts were temperature or saline treated, and 10 g samples were bioassayed in 16 mice and 12 cotton rats. No clinical sign or tissue infection was observed in these animals. Our study presents evidence that swine can be infected with C. bigenetica by ingesting oocysts present in snake feces or mouse feces (following inoculation of mice with snake-derived oocysts) or by ingesting C. bigenetica infected rodent tissue, that endogenously produced C. bigenetica oocysts are not excreted in the feces of swine, and that C. bigenetica in pork can be rendered noninfective by freezing at -20 degrees C (-4 degrees F) for 21 days, frying at 84 degrees C (183 degrees F) for 17 min, microwaving at 88 degrees C (190 degrees F) for 17 min, grilling at 82 degrees C (180 degrees F) for 48 min, baking at 95 degrees C (203 degrees F) for 230 min, boiling at 100 degrees C (212 degrees F) for 60 min, or by curing at 4 degrees C (39 degrees F) for 20 days.
1. Activity of S-adenosylmethionine decarboxylase, one of the rate-limiting enzymes of polyamine biosynthesis, was determined in oocysts of Eimeria stiedai, a coccidian parasite of the rabbit. 2. Several properties of the enzyme were compared to the mammalian enzyme. It showed considerably less substrate affinity than the analog enzyme from the rabbit. 3. The E. stiedai enzyme showed a low sensitivity to methylglyoxal bis(guanylhydrazone), a frequently used inhibitor of the enzyme in mammals, and two phenylated derivatives. 4. Results with the inhibitors are discussed in view of their potential use in chemotherapy.
Two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) have been identified from the parasitic protist Cryptosporidium parvum (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans including Plasmodium, Toxoplasma, and Eimeria possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.
Forms of the protozoan of the Hepatozoon genus are detected free in the circulation and also within some of the erythrocytes of infected snakes. In healthy snakes, DNA fragmentation and cell death usually affect a few circulating erythrocytes in agreement with the long life span expected for these cells. In the present study we investigated whether infection by Hepatozoon spp. affected the incidence of DNA fragmentation and cell death in erythrocytes from the rattlesnake, Crotalus durissus terrificus. Methods such as the kinetics of Feulgen-DNA hydrolysis, and the TUNEL and comet assays, previously used for the study of chromatin organization and DNA fragmentation in erythrocytes of healthy snakes, were used. The results indicated that Hepatozoon spp. increased the DNA fragmentation and chromatin condensation typical of cell death in circulating erythrocytes of C. d. terrificus, including cells that do not harbour the parasite. The Hepatozoon infection is thus suggested to accelerate destruction of erythrocytes in the rattlesnake, not only affecting cells harbouring the parasite, but also in those without it.
Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.
Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. bennetti was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collections.
The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.
Resistance to severe babesiosis in young calves has frequently been ascribed to an unknown serum factor(s) which inhibits growth of Babesia bovis in vitro. Our experiments show that young calf sera are as suitable as adult bovine sera for the in vitro culture of Babesia divergens, indicating that in this species at least inverse age resistance is due to alternative mechanisms. The suitability of commercial foetal calf sera for B. divergens cultures seems highly variable.
Parasite antigens are of interest both for the development of better diagnostic tools and potential subunit vaccines. Particularly, relevant in this regard are membrane proteins as the first point of contact between host and parasite. Here, four different methods for the extraction and solubilisation of Neospora caninum proteins were evaluated by comparing protein yield, specific antigenicity and relative protein abundance in the electrophoresis profile. Extraction with SDS and sulphobetaines (SB) gave higher yields than those achieved using the standard sonication method. SDS and SB preparations also gave the best signal to noise ratio when used as capture antigens in an ELISA. Electrophoresis of the three preparations showed an even protein-banding pattern spread out over the 90-10 kDa size range. However, there were a greater proportion of lower molecular weight proteins when the sonication method was used suggesting that this method may have resulted in the degradation/proteolysis of some proteins. Extraction with Triton X-114 resulted in the concentration of three or four proteins but led to a considerable reduction in the overall protein yield. The ELISA indicated that not all of the Triton X-114 extracted proteins were involved in specific antibody binding. The optimal extraction method for parasite proteins depends on the intended application.
Cryptosporidium molnari n. sp. is described from two teleost fish, the gilthead sea bream (Sparus aurata L.) and the European sea bass (Dicentrarchus labrax L.). The parasite was found mainly in the stomach epithelium and seldom in the intestine. Oocysts were almost spherical, with four naked sporozoites and a prominent residuum, and measured 3.23-5.45 x 3.02-5.04 (mean 4.72 x 4.47) microm in the type host, gilthead sea bream (shape index 1-1.17, mean 1.05). Sporulation was endogenous, as fully sporulated oocysts were found within the fish, both in the stomach epithelium and lumen, and in faeces. Oocysts and other stages of C. molnari fit most of the diagnostic features of the genus Cryptosporidium, but differ from hitherto described species, including piscine ones. All stages were located within a host contributed parasitophorous vacuole lined by a double host microvillar membrane. Merogonial and gamogonial stages appeared in the typical extracytoplasmic position, whereas oogonial and sporogonial stages were located deeply within the epithelium. Ultrastructural features, including the characteristic contact zone of the parasite with the host epithelial surface, were mostly coincident with those of other Cryptosporidium spp. Mitochondria were found in dividing meronts, merozoites, microgamonts and sporozoites. Pathological effects were more evident in gilthead sea bream, which also exhibited a clearly higher prevalence (24.4 versus 4.64% in sea bass). External clinical signs, consisting of whitish faeces, abdominal swelling and ascites, were rarely observed, in contrast with important histopathological damage. The wide zones of epithelium invaded by oogonial and sporogonial stages appeared necrotic, with abundant cell debris, and sloughing of epithelial cells, which detached to the lumen. No inflammation reaction was observed and the cellular reaction was limited to the cells involved in the engulfing of intraepithelial stages and debris, probably macrophages.
Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.