Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds.

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R (2), 0.98-0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 microM) and monensin (0.00225 to 0.144 microM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10(5) oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 microM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 microM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 microM or more, inhibition was at least 90%; 0.05 microM still yielded 80% inhibition, whereas at concentrations below 0.00625 microM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.

Immunoanalysis of three litters born to a Doberman bitch infected with Neospora caninum.

Neospora caninum is a tissue cyst-forming coccidium that may cause neuromuscular disorders in dogs. Infected bitches can transmit the parasite to their pups in utero. Vertical transmission may occur after primary infection during pregnancy and in subsequent pregnancies. The reason why only a few pups develop clinical neosporosis is unknown. We obtained sera from a Doberman bitch and its offspring delivered in three litters. The bitch had a titer of 1:640 in an indirect fluorescent antibody test (IFAT). At least three pups of litter A, one pup of litter B, and two pups of litter C were also seropositive for N. caninum. However, clinical neosporosis developed only in one pup of litter C, which had the highest IFAT titer (1:5,120) of all dogs examined. Western blots carried out after one-dimensional and two-dimensional separation of N. caninum tachyzoites revealed that the largest number of antigens was recognized by sera derived from the bitch. The lowest number of antigens was recognized by serum from the pup with clinical neosporosis. However, this pup uniquely recognized a major antigen with a molecular weight of about 17,000. The information collected in this study adds to our knowledge on why some pups develop clinical neosporosis and others do not.

Immunohistochemical and ultrastructural evidence for Neospora caninum tissue cysts in skeletal muscles of naturally infected dogs and cattle.

Protozoan stages were detected in the skeletal muscles of four dogs suffering from neosporosis and two neonatal calves with confirmed Neospora caninum- infection which could be immunohistochemically labelled by an antiserum against the bradyzoite-specific antigen BAG-5. In one calf, a tissue cyst was labelled by an antiserum against the N. caninum isolate NC-1. Ultrastructurally, a 0.3-1 microm-thick cyst wall surrounded the labelled parasites. The cysts were located within myofibres and contained varying numbers of bradyzoites each measuring 5.2(+/-0.6) x 1.6(+/-0.3) microm. The encysted stages showed typical ultrastructural features of N. caninum bradyzoites with subterminal nuclei, electron-dense rhoptries, and micronemes that were orientated perpendicular to the zoite pellicle. Immunohistochemistry and serology did not reveal any evidence for co-infection with Toxoplasma gondii. The detection of tissue cysts in skeletal muscle of N. caninum-infected intermediate hosts is of major epidemiological importance for the understanding of how definitive or intermediate carnivorous hosts become infected with N. caninum.

Toxoplasma gondii: from animals to humans.

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae.

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.

Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites.

Development and validation of species-specific nested PCRs for diagnosis of acute sarcocystiosis in sheep.

Sheep may be infected by four species of Sarcocystis. Two of these species, Sarcocystis tenella and Sarcocystis arieticanis, are pathogenic. They may cause abortion or acute disease during the early phase of infection, and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis in sheep has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic species. In addition, most of these tests show a reasonable sensitivity only for the late phase of infection. Therefore, diagnosis of acute sarcocystiosis has been based mainly on post-mortem examination, i.e. after the animal had succumbed to the disease. Here we established species-specific nested PCR assays based on unique small subunit ribosomal RNA gene sequences of S. tenella and S. arieticanis. These PCR assays specifically detect DNA of the homologous species in blood samples of sheep. No cross-reactions were observed with the heterologous pathogenic species, the non-pathogenic species Sarcocystis gigantea, or the closely related coccidia Toxoplasma gondii and Neospora caninum. In sheep experimentally infected with S. tenella or S. arieticanis, positive PCR results were correlated with the early phases of multiplication (endopolygeny) of the parasites. By contrast, Sarcocystis-specific antibodies were detected by an enzyme-linked immunosorbent assay only during the terminal phase of endopolygeny or thereafter. Thus, the nested PCR assays developed here enable, for the first time, the diagnosis and differentiation of infections with S. tenella and S. arieticanis in living sheep during the acute phase of the disease and facilitate comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.

Comparison of immunological and molecular methods for the diagnosis of infections with pathogenic Sarcocystis species in sheep.

Sheep may be infected by four species of Sarcocystis: Sarcocystis tenella and Sarcocystis arieticanis are pathogenic, Sarcocystis gigantea and Sarcocystis medusiformis are non-pathogenic. The two pathogenic species may cause abortion or acute disease during the early phase of infection and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic Sarcocystis species. In addition, most of these tests can only detect chronic sarcocystiosis. Therefore, diagnosis of acute sarcocystiosis or Sarcocystis-induced abortion has been based mainly on post-mortem examination, i. e. after the animal had succumbed to the disease. Recently, we have established species-specific PCR assays based on unique ribosomal RNA gene sequences of S. tenella and S. arieticanis. These assays enable the diagnosis and differentiation of infections with S. tenella and S. arieticanis in sheep intra vitam during the acute phase of the disease and, therefore, facilitate for the first time comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.