Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure

The characteristics of Myxobolus cuneus n. sp. and its relationship to the host Piaractus mesopotamicus are described based on light and electron microscopy and histological observations. Polysporic plasmodia measuring 20 microm to 2.1 mm in size were found in 63.3 % of the P. mesopotamicus examined. The parasite was found in the gall bladder, urinary bladder, gills, spleen, fins, head surface, liver and heart. Generative cells and disporoblastic pansporoblasts occurred along the periphery of the plasmodia, and mature spores were found in the internal region. The mature spores had a pear shaped body in frontal view, with a total length of 10.0 +/- 0.6 microm and a width of 5.1 +/- 0.3 microm (mean +/- SD). The spore wall was smooth with sutural folds. The polar capsules were elongated, were pear shaped, and equal in size (length 5.7 +/- 03 microm; width 1.7 +/- 0.2 microm), with the anterior ends close to each other. The polar filaments were tightly coiled in 8-9 turns perpendicular to the axis of the capsule. The plasmodia were always found in connective tissue (wall of the arterioles of the gill filaments, serous capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). The parasite caused important damage in the gills, where development occurred in the wall of gill filament arterioles; a mild macrophage infiltrate was also observed. In advanced developmental stages, the plasmodia caused deformation of the arteriole structure, with a reduction and, in some cases, obstruction of the lumen. The parasite was found throughout the period studied and its prevalence was unaffected by host size, season or water properties.     

鲫寄生碘泡虫属一新种

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Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

Unicapsula marquesi n. sp. (Mysxosporea, Multivalvulida) a gill parasite of Polydactylus quadrifilis (Cuvier, 1829) (Pisces, Polynemidae) from the senegalese coast (West Africa)]

Unicapsula marquesi n. sp. (Myxosporea) is described from gill filaments of Polydactylus quadrifilis (Pisces, Polynemidae) obtained from coats of Senegal. The cysts were elongated and their length was 1 to 3 mm. The spores were pyramidal and composed of three valves. Only one of theses valves contained a developed polar capsule measuring 3.01 +/- 0.09 microns in diameter. Length of spore was 6.13 +/- 0.21 microns and the width was 7.18 +/- 0.17. No filament like appendage at the extremity of shell valves. Data on ultrastructure of spores are presented.     

Internal ultrastructure of spores of microsporidans isolated from the Silkworm,Bombyx mori

Nosema bombycis, two Nosema spp., and a Pleistophora sp. were propagated in the silkworm and the fine structures of their spores were studied. The morphology of the polaroplast, the appearance of the nucleus, and the number of coils in the polar filament differed among the spores of the four species. The spores of the three Nosema species, however, had several identical components; e.g., the polaroplast was made up of two parts, they had two nuclei, and the ribosome arrangement was similar. On the other hand, the spore of Pleistophora sp. had a polaroplast composed of three parts, a single nucleus, and ribosomes arranged around the polar filament. Thus the fine structures of the spore differentiate microsporidan species.     

Spore dimensions of Puccinia species of cereal hosts as determined by image analysis

Digital image analysis was used to measure dimensions of spores produced by Puccinia coronata, P. graminis, P. hordei, P. recondita, P. striiformis and P. triticina. Included were teliospores, basidiospores, urediniospores and, except for P. striiformis, pycniospores and aeciospores. Length, width and projection area of spores were measured with NIH Image or Scion software. By using limits on size, spores were automatically selected and measured, except for teliospores, which required manual elimination of the pedicel and separation of images of adhering spores. Length and width were determined as the major and minor axes of the best fitting ellipse for each spore. This procedure gave values for length and width close to results obtained with an ocular micrometer. Projection area was determined as the number of pixels within spore boundaries multiplied by the area represented by each pixel, giving values that are not feasible to obtain accurately with an ocular micrometer. Of the species studied, spores of P. recondita had the largest dimensions, P. triticina had the smallest. The rank of the six species based on increasing width, length or projection area was almost the same, using each spore type except pycniospores. Generally, differences of 5% in a given spore dimension between two species were significant. Differences between species were greater with basidiospores and aeciospores than with other spore types. Teliospores were unique in that length and width were negatively correlated, resulting in less variation in area than in length or width. The results indicate that image analysis is useful for measuring spore dimensions, that projection area of spores is a useful added parameter for characterizing rust species and that dimensions of teliospores, basidiospores, aeciospores and urediniospores each are potentially useful for differentiating species.     

Multivalvulid myxozoans from eastern Australia: three new species of Kudoa from scombrid and labrid fishes of the Great Barrier Reef, Queensland, Australia

Three new species of Kudoa, each having 6 polar capsules, are described from the somatic muscle of fishes collected on the Great Barrier Reef, Queensland, Australia. Kudoa grammatorcyni n. sp. was observed in the shark mackerel Grammatorcynus bicarinatus. Spores are stellate in apical view, width (all measurements in microm) 8.62 (8.03-8.95); thickness 8.14 (7.63-8.68); suture width 7.7 (7.24-8.16); length 6.54 (6.32-6.71); polar capsule length 3.68 (3.55-3.82); polar capsule width 1.72 (1.65-1.84). Kudoa scomberomori n. sp. is described from the Spanish mackerel Scomberomorus commerson. Spores are stellate in apical view, width 7.56 (6.84-8.16); thickness 6.79 (6.18-7.63); suture width 5.92 (5.26-6.32); length 5.43 (5.00-6.18); polar capsule length 3.24 (3.03-3.55); polar capsule width 1.37 (1.25-1.51). Kudoa thalassomi n. sp. is described from the moon wrasse Thalassoma lunare. Spores are stellate in apical view, width 10.66 (9.47-11.84); thickness 9.37 (8.55-10.79); suture width 7.98 (6.84-8.82); length 6.65 (6.18-7.11); polar capsule length 4.92 (4.74-5.00); polar capsule width 2.12 (2.04-2.24). All 3 species differ in spore morphology from the 1 previously described myxozoan with 6 polar capsules, Hexacapsula neothunni from yellowfin tuna Neothunnus macropterus, which has since been reassigned to Kudoa.     

Block Staining of Nervous Tissue with Silver Studies of Fixatives, Lipid Solvents, and Reducing Solutions

Silver stains of the Cajal type, made on spinal cord of cats were studied to determine the limits of favorable ammonia concentration in alcohol as a fixative and the comparison of ammoniated alcohol with alcohol-chloroform and alcohol-pyridine mixtures. Such material subsequently extracted with various lipid solvents showed staining of a generally similar character. More intense staining was seen after the alkaline fixatives and best penetration of stain into the blocks after the most thoro extraction of lipids.

Experiments with reducing solutions which contained various proportions of pyrogallic acid and formalin indicated that pyrogallic acid is the essential ingredient.

Post-mortem autolysis up to 5 hours caused no change in fiber staining.     

Tuzetia boeckella sp. nov. (protozoa: Microsporida), a parasite of Boeckella triarticulata (Copepoda: Calanoidea) in Australia

A hitherto undescribed microsporidan has been found in the Australian freshwater copepod, Boeckella triarticulata, collected from Lake Burley Griffin, Canberra. We name this protozoan Tuzetia boeckella n. sp. and describe it in this paper. Large numbers of spores were found in the muscle of both sexes and all stages of the animals. The pyriform spores measured 5.1 × 2.7 μm with the extruded polar filament measuring 102 μm. Ultrastructural studies revealed the presence of a pansporoblastic membrane around each spore. The polar filament was arranged in a single row of 13–14 turns and decreased in diameter toward the posterior end. Few details of the life cycle were elucidated; however, evidence is presented for each sporont forming eight spores. Differentiating characters to distinguish this species from the six other known members of the genus are given.     

In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

Ultrastructural aspects of the myxosporean Henneguya astyanax n. sp. (Myxozoa: Myxobolidae), a parasite of the Amazonian teleost Astyanax keithi (Characidae)

This study reports light and electron microscopical aspects of a myxosporean found in the gills of the freshwater teleost Astyanax keithi Géry, Planquete & Le Bail, 1996 (family Characidae), collected from the estuarine region of the Amazon River, near Belém, Brazil. The prevalence of infection was 23%. In interlamellar spaces of the gills, ellipsoidal whitish cyst-like plasmodia structures were present, which contained spores. The spores had a spermatozoa-like appearance (47.8 +/- 0.71 microm in total length) with a fusiform body (15.2 +/- 0.77 pm in length, 5.7 +/- 0.71 microm in width and 4.2 +/- 0.31 microm in thickness), and each of the 2 valves presented a tapering tail (32.6 +/- 1.11 microm in length). The valves surrounded a binucleate sporoplasm cell and 2 polar capsules (5.0 +/- 0.13 microm in length, 1.5 +/- 0.07 microm in width) that contained 8 to 9 coils of the polar filament. In the sporoplasm, several unique sporoplasmosomes were visible. A synoptic table of spore measurements of known Brazilian Henneguya species is presented. The spores differed from those of previously described species. Based on spore morphology, it is concluded that this species belongs to the family Myxobolidae, genus Henneguya, and that it constitutes a new species: H. astyanax n. sp.     

Testicular myxosporidiasis in anurans,with a description of Myxobolus fallax n. sp.

During studies of amphibian sperm cryopreservation, a new species of myxosporidean parasite (Myxozoa, Myxosporae) was observed in the testes of the Australian dwarf green tree frog Litoria fallax (Peters). Myxosporidiasis was found to have no affect on L. fallax body condition or sperm numbers. Myxobolus spores from L. fallax are morphologically distinct from Myxobolus hylae spores (infecting the sympatric Litoria aurea Lesson) and the three previously named (exotic to Australia) Myxobolus species found in anurans. Myxobolus fallax n. sp. is characterised by: pseudocyst white, spherical to ovoid, 141 x 74 to 438 x 337 microm in diameter (mature); plasmodium with spores loosely arranged within interior. Spores ovoid 13.4 +/- 0.5 (12.6-14.6) microm length, 9.5 +/- 0.4 (8.3-10.6) microm width, 6.8 +/- 0.4 (6.5-7.6) microm depth, 1.4 +/- 0.1 (1.3-1.6) length/width; polar capsules broadly pyriform and equal in size 4.2 +/- 0.3 (3.3-4.7) microm length, 2.4 +/- 0.2 (2.1-2.8) microm width; filament coils 7-8, wound tightly and perpendicular to the longitudinal axis of the capsule; polar filament 34 +/- 7.0 (18-50) microm length; intercapsular appendix and sutural ridge folds absent; and iodinophilous vacuole and mucous envelope lacking. In addition to this new species, data from archival samples of M. hylae are provided which show two morphologically distinct spore types. Both appeared rarely in the same pseudocysts and we cautiously retain the single species.     

Ultrastructural studies of Henneguya rhamdia n. sp. (Myxozoa) a parasite from the Amazon teleost fish, Rhamdia quelen (Pimelodidae)

Henneguya rhamdia n. sp. is described in the gill filaments of the teleost fish Rhamdia quelen, collected from the Peixe Boi River, State of Pará, Brazil. This myxosporean produced spherical to ellipsoidal plasmodia, up to 300 microm in diameter, which contained developmental stages, including spores. Several dense bodies up to 2 microm in diameter were observed among the spores. The spore body was ellipsoidal (13.1 microm in length, 5.2 microm in width, and 2.5 microm in thickness) and each of the two valves presented a tapering tail (36.9 microm in length). These valves surrounded the binucleated sporoplasm cell and two equal ellipsoidal polar capsules (4.7 x 1.1 microm), which contained 10-11 (rarely 12) polar filament coils. The sporoplasm contained sporoplasmosomes with a laterally eccentric dense structure with a half-crescent section. Based on the data obtained by electron microscopy and on the host specificity, the spores differed from previously described Henneguya species, mainly in their shape and size, number and arrangement of the polar filament coils, and sporoplasmosome morphology.     

Block Staining of Nervous Tissue with Silver: II. Trichloracetic Acid, Sulfosalicylic Acid, Hofker's and Carnoy's Fluids as Fixatives

Alcoholic solutions of sulfosalicylic acid, trichloracetic acid and Hofker's solution fixed mammalian spinal cord with less shrinkage than ammoniated alcohol, but during the necessary alkalinization, washing, and silvering, the acid fixed specimens shrank more than those fixed in the alkaline alcohol. Specimens fixed in Carnoy's fluid shrank most. Successful silver stains were obtained after all the fixatives.     

Replication of Nosema Algerae In Three Insect Cell Lines

SYNOPSIS Nosema algerae , a microsporidan parasite of anopheline mosquitoes, was successfully replicated in 3 insect cell culture lines: Trichoplusia ni (TN-368); Heliothis zea (IPLB-1075); and Mamestra brassicae (IZD-Mb-0503). Infectious spores were produced in vitro. Spores were observed at 48 h postinfection, and some cells were filled with sproes by 72 h.
The number of parasites per cell increased with time. At 72 h postinfection, the infection rates for the 3 cell lines ranged from 23 to 32%. Infected cell lines were subcultured, and by the 6th passage spore production had ceased.     

A new species of Myxozoa, Henneguya rondoni n. sp. (Myxozoa), from the peripheral nervous system of the Amazonian fish, Gymnorhamphichthys rondoni (Teleostei)

Henneguya rondoni n. sp. found in the peripheral lateral nerves located below the two lateral lines of the fish Gymnorhamphichthys rondoni (Teleostei, Rhamphichthyidae) from the Amazon river is described using light and electron microscopy. Spherical to ellipsoid cysts measuring up to 110 microm in length contained only immature and mature spores located in close contact with the myelin sheaths of the nervous fibres. Ellipsoidal spores measured 17.7 (16.9-18.1)-microm long, 3.6 (3.0-3.9)-microm wide, and 2.5 (2.2-2.8)-microm (n=25) thick. The spore body measuring 7.0 (6.8-7.3)-microm long was formed by two equal symmetric valves, each with an equal tapering tail 10.7 (10.3-11.0) microm in length. The tails were composed of an internal dense material surrounded by an external homogeneous sheath of hyaline substance. The valves surrounded two equal pyriform polar capsules measuring 2.5 (2.2-2.8)-microm long and 0.85 (0.79-0.88)-microm (n=25) wide and a binucleated sporoplasm cell containing globular sporoplasmosomes 0.38 (0.33-0.42) microm (n=25) in diam. with an internal eccentric dense structure with half-crescent section. Each polar capsule contains an anisofilar polar filament with 6-7 turns obliquely to the long axis. The matrix of the polar capsule was dense and the wall filled with a hyaline substance. The spores differed from those of previously described species. Based on the ultrastructural morphology of the spore and specificity to the host species, we propose a new species name H. rondoni n. sp.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

Henneguya episclera sp. n. (Protozoa: Myxosporida), a new myxosporidan from the eye of the pumpkinseed sunfish.

Henneguya episclera sp. n. is described from the episclera of the eye of the pumpkinseed sunfish (Lepomis gibbosus). Mean spore dimensions (in micrometers) are: total length 62.6; spore length 21.7; spore thickness 8.0; spore breadth 8.7; tail lengths 37.1 and 40.9; polar capsule lengths 6.0 and 6.5; polar capsule breadths 2.7 and 3.0; polar capsule thickness 3.5; and vacuole 5 by 5.     

Effects of Various Fixing and Storage Fluids on Starch in Sapwood

Wood material that is to be used for observation of starch content may be stored many months in several plant material fixatives, formalin-acetic-alcohol, formalin-propionic-alcohol, tertiary butyl alcohol, ethyl alcohol, and 5 and 10% aqueous formalin without any apparent deleterious effect on the starch granules Storage of wood in aqueous formalin solutions of 15% and higher concentrations caused starch granules to break down and disperse forming a coagulated mass, in which it was impossible to see the individual grains.