Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.

Coccidian parasites of fish encompass profound phylogenetic diversity and gave rise to each of the major parasitic groups in terrestrial vertebrates.

Fish are the oldest and most diverse group of vertebrates; it therefore stands to reason that fish may have been the original hosts for many types of extant vertebrate parasites. Here, we sought to determine whether coccidian parasites of fish are especially diverse. We therefore sampled such parasites from thirty-nine species of fish and tested phylogenetic hypotheses concerning their relationships, using 18S rDNA. We found compelling phylogenetic support for distinctions among at least four lineages of piscine parasites presently ascribed to the genus Goussia. Some, but not all parasites attributed to Eimeria were confirmed as such. Major taxonomic revisions are likely justified for these parasites of fish, which appear to have given rise to each of the major lineages of coccidian parasites that subsequently proliferated in terrestrial vertebrates, including those such as Toxoplasma gondii that form tissue cysts in intermediate hosts.

N-Glycosylation analysis of formalin fixed paraffin embedded samples by capillary electrophoresis.

In this study, N-linked glycans from intact, formalin treated and formalin fixed paraffin embedded (FFPE) standard glycoproteins, human serum and mouse tumor tissue samples were investigated in respect to their susceptibility for formaldehyde treatment mediated changes. FFPE samples were first deparaffinized, followed by solubilization in radioimmunoprecipitation assay buffer and treated with PNGase F for N-glycan release. The released glycans were labeled with a charged fluorophore and analyzed by capillary electrophoresis with laser induced fluorescent detection. No significant alterations were found in the N-glycome profile at any of the investigated complexation levels (i.e., glycoprotein, serum and tissue samples) of the study. These results suggest that FFPE samples can be readily used for global N-glycome analysis holding the promise to find novel carbohydrate biomarkers in prospective and retrospective studies. Exoglycosidase based carbohydrate sequencing was also applied to reveal some basic structural information about the N-linked carbohydrates of the mouse tumor tissue samples.

Eimeria that infect fish are diverse and are related to, but distinct from, those that infect terrestrial vertebrates.

The Eimeria are ubiquitous parasites (Phylum: Apicomplexa; family: Coccidia) of the gut epithelium of vertebrates which complete their development in a single host species and whose sporocysts may be recognized by the presence of a Stieda body through which their sporozoites excyst. Their diversity and relationship to other kinds of coccidia have been successfully explored by molecular systematic studies based on the sequencing the 18S ribosomal DNA. To date, most attention has been paid to the diversity and evolutionary relationships of Eimeria spp. parasitizing terrestrial vertebrates, most especially those species infecting domesticated birds and mammals. Regrettably, no Eimeria have yet been considered from the Earth's first vertebrates: the fish. If Eimeria first evolved in fish, then extant piscine parasites should comprise a deeply branching assemblage at the base of well-constructed phylogenetic trees. Here, we sequenced portions of ribosomal DNA from several such isolates (from Eimeria anguillae, Eimeria daviesae, Eimeria percae, Eimeria variabilis, Eimeria rutili and Eimeria nemethi) and compared them to one another as well as to other available sequences from the parasites of fish and terrestrial vertebrates, in order to better understand their diversity and origins. By establishing that such piscine parasites comprise a deeply branching clade at the base of the Eimeriidae, these data substantiate the hypothesis that Eimeria may have originated in fish. Plainly, a great deal of coccidian diversity awaits future discovery and description.

Myxozoan infection of the Malaysian mahseer, Tor tambroides, of Tasik Kenyir Reservoir, Malaysia: description of a new species Myxobolus tambroides sp.n.

Tor tambroides, a common and appreciated cyprinid fish of the Tasik Kenyir water reservoir in Malaysia, is one of the species selected for propagation. This fish was first successfully propagated in Malaysia by the Department of Agriculture, Sarawak, Malaysia, and the breeding program continued throughout the country. The gills were frequently infected by a Myxobolus species to be described as Myxobolus tambroides sp. n. The small, 50 to 70 µm, round plasmodia of this species is located intralamellarly. Plasmodia were filled with pyriform myxospores, 9.9 and 7.4 µm wide. In sutural view, the caudal end of the myxospores had a distinctive valvular groove, parallel with the suture. Plasmodia caused deformations on the affected and the neighbouring gill lamellae. The 18S rDNA sequence of M. tambroides sp.n. did not show a close relationship with any other Myxobolus spp., represented in the GenBank. This might be an emerging parasite likely to impact the propagation of this fish.

Efficacy of some anticoccidial drugs for treating coccidial enteritis of the common carp caused by Goussia carpelli (Apicomplexa: Eimeriidae).

In this study, nine anticoccidial drugs commonly used in poultry were tested for efficacy for the prevention and treatment of Goussia carpelli (Apicomplexa) infection in common carp (Cyprinus carpio L.). To establish experimental infection with G. carpelli, paratenic host oligochaetes of the genera Tubifex and Limnodrilus were infected with oocysts, and laboratory-cultured parasite-free common carp fingerlings were infected by feeding to them oligochaetes containing sporozoites. The anticoccidial drugs (amprolium, narasin, maduramicin, salinomycin Na, lasalocid Na, diclazuril, robenidine HCl, monensin Na and toltrazuril), mixed in the food of the fish in a dose of 200 mg/kg, were fed for 12 days. Common carp fingerlings fed diclazuril, lasalocid, robenidine HCl or maduramicin and killed on day 14 after exposure were free from infection, while other groups treated with amprolium, toltrazuril, monensin Na, narasin or salinomycin Na harboured oocysts in the mucus and epithelium of the gut.