Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

Construction of PCR primers to detect and distinguish Eimeria spp. in northern bobwhites and a survey of Eimeria on gamebird farms in the United States.

Coccidiosis is an important disease in captive gamebirds, including northern bobwhites (Colinusvirginianus). Three Eimeria species, Eimeria lettyae, Eimeria dispersa, and Eimeria colini, have been described in bobwhites. Distinguishing the various Eimeria spp. is often problematic because of similarity in oocyst morphology and site of infection and thus requires live bird infections to distinguish between the coccidian species. To aid in identification and diagnosis, PCR specific primers were generated against the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene using sequences obtained from coccidian-positive samples collected from diagnostic cases or litter from captive bobwhites. Three distinct Eimeria spp. were detected. Species-specific primers were constructed and used to survey the prevalence of the species in 31 samples collected from 13 states. The primers survey results identified E. lettyae, E. dispersa, and Eimeria sp. in 20 (64.5%), 22 (71%), and 29 (93.5%) of the samples, respectively. Mixed infections were common: 13 (41.9%) samples had 3 Eimeria spp., 14 (45.2%) had 2 spp., and 4 (12.9%) samples had only 1 species. The species were widely distributed over the area sampled and were not associated with the age of the flock.

Partial sequence of the alpha-tubulin gene from Histomonas meleagridis isolates from the United States.

Histomonas meleagridis , the causative agent of histomoniasis, is a protozoan parasite classified in the Dientamoebidae (order Tritrichomonadida). The α-tubulin gene of 7 H. meleagridis isolates originating from either domestic chickens or turkeys from the United States was amplified by nested PCR and sequenced. A 91.4-99.8% nucleotide identity was shared among the 7 different sequences, and phylogenetic analysis disclosed that the 7 isolates were divided into at least 3 clades. These sequences had a 91-99% nucleotide identity and a 96-100% amino acid identity compared with 3 H. meleagridis α-tubulin sequences obtained from isolates originating from turkeys in Germany. Further α-tubulin gene analysis from species in the Dientamoebidae will be useful in elucidating the evolutionary relationship of these protozoans.

Molecular characterization of Histomonas meleagridis and other parabasalids in the United States using the 5.8S, ITS-1, and ITS-2 rRNA regions.

Extracted DNA from 28 Histomonas meleagridis -infected avian tissue samples from multiple hosts and geographic locations was analyzed for variation in the 5.8S rRNA and the flanking internal transcribed spacer regions (ITS 1 and ITS 2). Samples were amplified by polymerase chain reaction, sequenced, and compared with known sequences from GenBank accessions of H. meleagridis and other related protozoa. The analyses revealed significant genetic variation within H. meleagridis sequences and suggested the possibility of multiple genotypes within the samples or a possible misdiagnosis. Related protozoa found in some samples were mostly identified as Tetratrichomonas spp. However, 1 sample had a 93% identity to Simplicimonas similis , a newly described organism, suggesting the possibility of a new pathogen in poultry. A phylogenetic tree analyzing the 5.8S and flanking ITS regions was inconclusive and we were unable to resolve all H. meleagridis into a single grouping. In contrast, a tree constructed only on the 5.8S rRNA grouped all but 1 H. meleagridis sample into 1 clade, including GenBank accessions submitted from Europe. This suggests that the 5.8S region alone is more reliable in identifying H. meleagridis than are the combined 5.8S and flanking ITS regions. There was no correlation between genotypes and host species or geographic location, suggesting that H. meleagridis moves freely between multiple avian species in the sampled regions.

Oocyst production of Eimeria lettyae in northern bobwhites following low-dose inoculations.

Inoculation of northern bobwhite quail ( Colinus virginianus ) with low doses of Eimeria lettyae oocysts stimulates a protective immune response, suggesting immunization may be an option for controlling coccidiosis. However, the oocyst production of inoculated birds could be considerable, leading to subsequent outbreaks. To determine the oocyst production following inoculation with E. lettyae, we orally infected 12-wk-old bobwhites with 100, 1,000, or 10,000 sporulated oocysts. Fecal materials were collected on days 5-9 post-inoculation, and total oocyst production was counted in McMaster chambers. Oocyst production/bird was 49.75, 89.5, and 436 × 10(6) for 100, 1,000, or 10,000 oocysts administered, respectively. Estimated oocysts produced/oocyst administered was 49.75, 8.95, and 4.36 × 10(4) for 100, 1,000, or 10,000 oocysts administered, respectively. These findings not only illustrate the crowding effect of larger oocyst inocula but also illustrate the fecundity of E. lettyae at low doses. This suggests that successful immunization of bobwhites against coccidiosis with live vaccines might require attenuated strains with reduced reproductive potential.

Parasitic ventriculitis caused by Hadjelia truncata in California rock pigeons (Columba livia).

Severe ventriculitis and emaciation caused by the infestation of the nematode Hadjelia truncata occurred in meat-type breeder rock pigeons (Columba livia) in southern and central California. Hadjelia truncata can infest several species of birds, although it has only been reported as pathogenic in pigeons. The factors that contribute to H. truncata pathogenicity are not known. The gross and microscopic pathology caused by the infestation of H. truncata in the ventriculus of pigeons and its morphological identification are presented.

Biochemical characterization of enoyl reductase involved in Type II fatty acid synthesis in the intestinal coccidium Eimeria tenella (Phylum Apicomplexa).

An enoyl reductase (EtENR) closely related to those of green algae and involved in Type II fatty acid synthesis was characterized and localized to the apicoplast in the coccidium Eimeria tenella. Biochemical analysis using native EtENR protein extracted from parasites confirmed its function as an enoyl reductase using NADH as a cofactor. However, the recombinant form (rEtENR) expressed in bacteria was only able to oxidize NADH, but unable to transfer the electron to enoyl-CoA, possibly due to the inappropriate folding of rEtENR expressed in bacteria. The functions of both native and recombinant EtENR could be inhibited by triclosan (IC(50)=1.45 microM), suggesting that this enzyme may be explored as a drug target against coccidiosis.

High yield of parasites and prolonged in vitro culture of Histomonas meleagridis.

Dwyer's medium is a frequently employed culture medium for Histomonas meleagridis with rice powder as an essential ingredient. The effect of adding larger quantities of rice powder to the culture medium and the influence of the size of the rice particles on the growth of H. meleagridis was examined. Increasing the amount of rice powder from the standard amount of 10 to 12 mg to 50 to 100 mg per 12.5 ml medium resulted in approximately a 10-fold increase of parasites. Larger quantities of rice powder did not give better yields. The particle size of the rice powder proved relatively unimportant, although the addition of only large rice powder particles (>250 microm) resulted in a somewhat lesser yield. H. meleagridis cultures could be prolonged from approximately 4 days to at least 2 weeks without subculturing by supplementing the culture medium with rice powder only.

Apicoplast genome of the coccidian Eimeria tenella.

Unicellular apicomplexans possess an algal-originated plastid referred to as an apicoplast. Although apicomplexan parasites are comprised of highly diverse protists, the complete apicoplast genome sequences have only been determined from the hematozoan Plasmodium falciparum and cyst-forming coccidian Toxoplasma gondii. Here, we report the third complete sequence of apicoplast genome from the intestinal coccidian Eimeria tenella that may serve as a new drug target against coccidiosis in the livestock. The AT-rich E. tenella plastid genome is a 35-kb circular element. Its gene organization resembles more closely that of T. gondii than P. falciparum. Although the E. tenella plastid genome contains an almost identical set of genes to that found in P. falciparum and T. gondii, its encoded genes share low or moderate homologies with their counterparts in the other two apicomplexans. With the addition of this coccidian plastid genome sequence, we attempted to reexamine the apicoplast genome evolution and performed phylogenetic reconstructions using maximum likelihood and Bayesian inference (BI) methods based on a concatenated dataset of plastid-encoded rpoB, rpoC1 and rpoC2 proteins. All resulting rpo protein trees placed apicoplast as a sister to Euglena within the green lineage. On the other hand, many recent studies based on the organization of plastid genes and some nuclear-encoded plastid proteins have supported a common red algal ancestry of apicomplexan and dinoflagellate plastids. If the apicoplast indeed originated from a red ancestor, the green relationship of apicomplexan genes would probably imply that the ancestral host that gave rise to the (red) apicoplast might have already contained some primary green plastid genes.