Survey and genetic characterization of wastewater in Tunisia for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp.

The microbial diversity of wastewater used for irrigation and fertilization was assessed using specific polymerase chain reaction (PCR) assays to detect and genotype several pathogenic protists including Cryptosporidium spp., Giardia duodenalis, Cyclospora spp., Eimeria spp. and Enterocytozoon bieneusi. A total of 220 wastewater samples (110 raw, 110 treated) and 12 sludge samples were collected from 2005 to 2008 from 18 treatment plants located throughout Tunisia. Except for Cyclospora, which was detected only once, E. bieneusi (61%), G. duodenalis (28%), Cryptosporidium spp. (27%) and Eimeria spp. (45%) were frequently observed in wastewater and sludge. Sequencing of PCR products showed that C. hominis, C. andersoni, G. duodenalis sub-assemblage A-II and E. bieneusi genotypes D and IV were the most prevalent. An analysis of the distribution of 209 internal transcribed spacer sequences of E. bieneusi originating from wastewater at the 18 treatment plants showed a similar genetic diversity, regardless of the geographical location. The identification of these parasite species and genotypes and of host-specific Eimeria species indicates that the microbial quality of wastewater was impacted by humans, livestock and rodents. Given the public health risks that some of these parasites represent, guidelines on wastewater usage are needed to minimize human exposure to these pathogens.

Dynamics of transcription of immunomodulatory genes in endothelial cells infected with different coccidian parasites.

Sporozoites of Eimeria bovis and tachyzoites of Neospora caninum and Toxoplasma gondii are able to invade and to replicate in endothelial cells. Here we report on responses of bovine umbilical vein endothelial cells (BUVEC) in vitro to these coccidial infections by determining mRNA levels of the CXC chemokines GRO-alpha, IL-8 and IP-10, the CC chemokines MCP-1 and RANTES and of GM-CSF, COX-2 and iNOS relative to the level of housekeeping gene (GAPDH) transcription. T. gondii and N. caninum tachyzoites caused profound transcriptional upregulation of all genes in question. In general, upregulation started 2-4 h p.i. and maximum transcript levels were observed 4 h p.i. GRO-alpha and IL-8 gene transcription had decreased to almost control levels by 12 h p.i.; in the case of the other chemokines enhanced transcript levels persisted longer or showed a biphasic time-course. A similar time-course to CC chemokines was observed for GM-CSF mRNA, whilst COX-2 gene transcript peaks were detected at 2-4 h p.i. and 48-72 h p.i. iNOS mRNA levels increased from 4 to 48 h p.i. In contrast, E. bovis sporozoites failed to induce the transcription of CXC chemokine genes and of COX-2, and only caused moderate transcription upregulation of the other genes considered. In conclusion, infections of BUVEC with these coccidian parasites result in host cell activation associated with enhanced transcription of genes encoding for proinflammatory and immunomodulatory molecules, which are important for innate immune reactions and the transition to adaptive immunity. Differences between E. bovis versus T. gondii and N. caninum may illustrate a particular evasion strategy of E. bovis sporozoites, which is related to their need to persist in the host cell for a long period of time and to the avoidance of inflammatory process-induction.

Neospora caninum and Hammondia heydorni are separate species/organisms.

Neospora caninum and Hammondia heydorni are two coccidian parasites with morphologically similar oocysts in canine feces. It was recently proposed that they are one species. In this paper, we review the biology and morphology of these parasites and present evidence that N. caninum and H. heydorni are separate species.

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.

Free-Tree--freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of genus Frenkelia.

The Win95 program for computation of distance matrixes and construction of phylogenetic or phenetic trees on the basis of RAPD, RFLP and allozyme data was presented. In contrast with other presently available software, the program FreeTree can also assess the robustness of the tree topology by bootstrap, jackknife or OTU-jackknife analysis. Moreover, the program can be used also for an analysis of data obtained in several independent experiments performed with nonidentical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 22 strains of Frenkelia. The program is available as an autoextractive archive containing the installation files of FreeTree and TreeView, manual in MS Word format and a sample of the input file at http://www.natur.cuni.cz/flegr/programs/+ ++freetree.

Codon usage and bias among individual genes of the coccidia and piroplasms.

Codon usage has been analysed in individual gene sequences, derived from a variety of parasitic protozoa in the class Sporozoa of the phylum Apicomplexa using metric multidimensional scaling. The two groups of codon usage patterns detected reflect the two main subgroups of organisms studied (the coccidia and the piroplasms), and it is the pattern of usage of synonymous codons that has the largest influence on overall codon usage in the individual genes, rather than being the pattern of amino acid composition of the gene product. The magnitude of the codon usage bias in the sequences was determined using three commonly used indices-NC, GC3S and B. In general, although relatively low levels of codon usage bias were detected in these gene sequences, codon usage bias does explain at least some of the codon usage patterns observed. Codon usage bias was observed to be dependent on the overall base composition of the genes analysed, which in turn was reflected in the types of codons that were either over- or under-represented in the nucleotide sequences. In keeping with observations on prokaryotic organisms, it is speculated that the codon usage patterns detected in these parasitic protozoa are the result of directional mutation pressure on the base composition of the genomic DNA.

The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence.

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

The genus Hammondia is paraphyletic.

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

Phylogenetic analysis of coccidia based on 18S rDNA sequence comparison indicates that Isospora is most closely related to Toxoplasma and Neospora.

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa, continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis, and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.