Levels of infection, pathology and nodule size of Onchocerca flexuosa (Nematoda: Onchocercidae) in red deer (Cervus elaphus) from northern Spain.

Between 2005 and 2007, the presence of Onchocerca flexuosa (Wedl, 1856) was discovered and investigated in 110 red deer (Cervus elaphus) shot in the Riaño Regional Hunting Reserve, in the province of León (north-western Spain). Nodules containing O. flexuosa were located in the dorsal region and flanks of the deer. These were collected and measured, and some adult parasites were extracted from the nodules and identified by morphology and by obtaining mitochondrial 12S rDNA sequences, which were identical to those of previously published sequences for O. flexuosa. Some nodules were prepared for histology, embedded in paraffin, sectioned and stained with haematoxylin-eosin. Histologically, the worms were found in several compartments separated by an infiltrated fibrous tissue. These compartments were inhabited by several females and males, surrounded by a fibrous capsule. A total of 85.45% (95% confidence interval (CI): 78.86-92.04%) of red deer were parasitized, with a mean intensity of 9.53 ± 12.27 nodules/host, ranging between 1 and 74 nodules/deer. Significant differences in prevalence and intensity of infection were found between young and adult red deer, and also between seasons. However, no significant differences between males and females were observed. Five hundred and ninety-seven nodules were measured (15.81 ± 3.94 mm) and classified by sizes into small ( < 10 mm), medium (10-20 mm) and large (>20 mm). No relation was found between the size of the nodules and the time of infection. The high values found in the studied parameters show that northern Spain is an area of high-intensity infection for deer.

A two-phase case-control study for colorectal cancer genetic susceptibility: candidate genes from chromosomal regions 9q22 and 3q22.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts.

The genus Atoxoplasma (Garnham 1950) as a junior objective synonym of the genus Isospora (Schneider 1881) species infecting birds and resurrection of Cystoisospora (Frenkel 1977) as the correct genus for Isospora species infecting mammals.

Molecular and morphological data permit a rational subdivision of the paraphyletic Isospora into 2 apparently monophyletic groups of parasites, i.e., Isospora and Cystoisospora. Atoxoplasma was determined to be a junior objective synonym for Isospora. Tetrasporozoic, diplosporocystic oocysts possessing Stieda bodies in their sporocysts belong to Isospora (Eimeriidae) and have been described principally from the feces of birds. Tetrasporozoic, diplosporocystic oocysts without Stieda bodies in their sporocysts belong to Cystoisospora (Sarcocystidae).

PCR-based quantitation of Cryptosporidium parvum in municipal water samples.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.

Small subunit ribosomal RNA genes of tabanids and hippoboscids (Diptera: Brachycera): evolutionary relationships and comparison with other Diptera.

The small subunit ribosomal RNA (SSU rRNA) genes of hippoboscid (Ornithoica vicina Walker) and tabanid (Chrysops niger Macquart) Diptera were sequenced to determine their phylogenetic position within the order and to determine whether or not extensive hypervariable regions in this gene are widespread in the Diptera. A parsimony analysis of an alignment containing 8 dipteran sequences produced a single most parsimonious tree that placed O. vicina as sister group to Drosophila melanogaster Meigen. The tabanid Chrysops niger was sister group to the asilomorphan taxa, and the sister group to the Brachycera was a Tipula sp. although this relationship was not supported by bootstrap analysis. The hippoboscid and tabanid sequences contain extensive hypervariable regions in the V2, V4, V6, and V7 regions as do other Diptera. When these regions of the alignment were excluded from the phylogenetic analysis, a single most parsimonious tree was found. This tree had an identical overall topology to the tree obtained from the total data set. The hypervariable regions in parts of the dipteran SSU rRNA genes were more extensive in the nematocerous dipteran sequences used in this study than in the other dipteran representatives; these hypervariable regions may be of more utility in inferring relationship among species and subspecies than at the suprageneric level.

Observability in dynamic evolutionary models.

In the paper observability problems are considered in basic dynamic evolutionary models for sexual and asexual populations. Observability means that from the (partial) knowledge of certain phenotypic characteristics the whole evolutionary process can be uniquely recovered. Sufficient conditions are given to guarantee observability for both sexual and asexual populations near an evolutionarily stable state.

Observability in strategic models of viability selection.

Strategic models of frequency-dependent viability selection, in terms of mathematical systems theory, are considered as a dynamic observation system. Using a general sufficient condition for observability of nonlinear systems with invariant manifold, it is studied whether, observing certain phenotypic characteristics of the population, the development of its genetic state can be recovered, at least near equilibrium.

Hemangiomas, vascular malformations, and lymphovenous malformations: classification and methods of treatment.

A total of 207 patients with hemangiomas, vascular malformations, and lymphovenous malformations were treated by the same surgeon from 1980 to 1990. Thirty-seven patients with true hemangiomas underwent surgical treatment. Only those hemangiomas which caused functional or developmental disturbances or those with complications were treated; many more were allowed to regress spontaneously. Sixty-five patients with low-flow and 16 with high-flow vascular malformations were treated by using a variety of surgical approaches. In low-flow lesions, sclerosant therapy can be extremely effective, either alone, in small lesions, or combined with surgical resection or embolization, in larger lesions. Preoperative embolization and surgical excision are the treatment of choice in high-flow malformations. Twenty-seven patients with lymphovenous malformations had only surgical excision with a high success rate. Sixty-two patients with acquired "senile hemangiomas" underwent a single local excision with excellent results. When indicated, angiography has been of great value as a diagnostic procedure to provide information about the vascular dynamics and the extent of these lesions, although magnetic resonance imaging is now being used more frequently for this purpose. Selective angiography also was used as a therapeutic modality when embolization was part of the treatment protocol. A new classification based on clinical, histologic, and vascular flow characteristics of these lesions has been used to simplify the present nomenclature and to help in selection of the most appropriate treatment. It has the added value of being in the language of the radiologist, who should be a member of the vascular anomalies team.

Evolutionary relationships among the protostrongylidae (Nematoda: Metastrongyloidea) as inferred from morphological characters, with consideration of parasite-host coevolution.

The phylogeny of nematodes in the family Protostrongylidae (Nematoda: Metastrongyloidea) was reconstructed by cladistic analysis of 28 binary and multistate characters derived from comparative morphology. Analyses were hierarchical, and examined (1) relationships among genera, including 13 ingroup taxa and Metastrongylidae as an outgroup (single tree, 78 steps, consistency index [CI] = 0.705); and (2) relationships among genera and species groups, including 21 ingroup taxa and Metastrongylus apri as an outgroup (single tree, 76 steps, CI = 0.582). In the species-level tree, Protostrongylidae was divided into 2 major clades, 1 containing the subfamilies Muelleriinae (including the recently described Umingmtakstrangylus pallikuukensis), Elaphostrongylinae, and the Varestrongylinae (excluding Pneumocaulus kadenazii). Varestrongylus was paraphyletic as it included Pneumostrongylus calcaratus. The second major clade consisted of a paraphyletic group containing Protostrongylus spp. and Spiculocaulus leuckarti and, basal to this subclade, several other individual protostrongylid lineages. The various subclades generally correspond to the subfamilial divisions of the Protostrongylidae. The Neostrongylinae, however, is not supported as Neostrongylus and Orthostrongylus are not sister groups. Based on a large number of hypothesized synapomorphies, the elaphostrongylines appear to be a highly derived group of protostrongylids, a feature potentially correlated with their habitat localization in muscular and nervous tissues. The generic-level tree retained most of the primary structure revealed among the species but excluded the varestrongylines from the Muelleriinae + Elaphostrongylinae subclade. Artiodactyles of the family Cervidae are considered basal hosts for protostrongylids; secondary colonization in Caprini, Rupicaprini, and among lagomorphs is postulated.

An Eimeriid origin of isosporoid coccidia with Stieda bodies as shown by phylogenetic analysis of small subunit ribosomal RNA gene sequences.

Morphological and life cycle features of the tissue cyst-forming coccidia have been difficult to interpret in devising taxonomic classifications for the various genera. In this study, we amplified the full small subunit rRNA gene sequence of Isospora robini McQuistion and Holmes, 1988, and the partial sequence of Isospora gryphoni Olsen, Gissing, Barta, and Middleton, 1998 by PCR. Both of these species vary from Isospora species of mammals in having Stieda bodies on the sporocysts. The sequences were cloned and sequenced and were incorporated into an alignment with other Isospora species lacking Stieda bodies as well as with other coccidia. Maximum parsimony analysis of these sequences produced a single most parsimonious tree that placed I. robini and I. gryphoni in a clade containing various other eimeriid species. The Isospora species lacking Stieda bodies were in the sarcocystid clade. Similar results were found by maximum likelihood analysis. These findings indicate that the genus Isospora as defined by several authors is polyphyletic. Taxonomic changes to the genus Isospora would have to incorporate the 2 major clades found by molecular phylogenetic analysis. Isospora species with Stieda bodies should be classified in the family Eimeriidae, whereas those without Stieda bodies should remain in the family Sarcocystidae.

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.

Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora.

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.

DNA fingerprinting of Cryptosporidium parvum isolates using amplified fragment length polymorphism (AFLP).

The genetic variability of 10 Cryptosporidium parvum isolates of human and animal origin was investigated using amplified fragment length polymorphism (AFLP). Analysis of fluorescent dye-labeled amplified products was carried out using an ABI PRISMS 377 DNA sequencer and ABI PRISMS GeneScan software. One-hundred and twelve primer combinations were evaluated using a single C. parvum isolate. The patterns generated were highly reproducible. For subsequent study, a subset of 9 primer pairs that yielded 30-90 DNA fragments after the polymerase chain reaction, within the size range of 50-500 bp, was used to screen the 10 C. parvum isolates, including 7 bovine, 1 equine, and 2 of human origin. The animal isolates produced identical fingerprint patterns with every primer combination tested. Of the 2 human isolates tested, 1 of the isolates, passaged in calves, generated the same AFLP DNA banding patterns as the animal isolates, whereas the other isolate, obtained directly from human feces, produced unique patterns. Polymorphism, detected by comparison of the fingerprint patterns of the latter human isolate with the common pattern shared by all other isolates, ranged from 17 to 35% for the 9 primer pairs. The results show that AFLP is a useful method for differentiating C. parvum isolates into 2 distinct genotypes.

Cerebral cysticercosis in a woodchuck (Marmota monax).

A juvenile woodchuck (Marmota monax) with vestibular signs was found in Woodbridge, Ontario (Canada) and later euthanized. At necropsy there was marked distortion of the right side of the skull, where a large, fluctuant, subcutaneous mass extended under the zygomatic arch and caudally from the right eye towards the right ear. The mass was multiloculated and contained a large number of tapeworm cysticerci, each about 1 to 2 mm in diameter. The third and lateral ventricles of the brain were dilated and contained large numbers of similar cysticerci. Based on the exogenous budding of cysts and the morphology of the scolex in each cyst, they were identified as cysticerci of Taenia crassiceps. This is the first report of cerebral cysticercosis in a woodchuck.

Complement component C3 mediates Th1/Th17 polarization in human T-cell activation and cutaneous GVHD.

The complement system has been shown to regulate T-cell activation and alloimmune responses in GVHD. Mice deficient in the central component of complement system C3 have significantly lower GVHD-related mortality/morbidity, and C3 modulates Th1/Th17 polarization in mouse GVHD. To investigate whether anticomplement therapy has any impact on human T-cell activation, a drug candidate Compstatin was used to inhibit C3 activation in this study. We found the frequency of IFN-γ (Th1)-, IL-4 (Th2)-, IL-17 (Th17)-, IL-2- and TNF-α-producing cells were significantly reduced among activated CD4(+) cells in the presence of Compstatin. Compstatin treatment decreased the proliferation of both CD4(+) and CD8(+) T cells upon TCR stimulation. However, Compstatin does not affect the production of IL-2 and TNF-α in activated CD8(+) T cells, and the differentiation of CD8(+) T cells into distinct memory and effector subsets remained intact. Furthermore, we examined complement deposition in skin and lip biopsy samples of patients diagnosed with cutaneous GVHD. C3 deposition was detected in the squamous epithelium and dermis, blood vessels and damaged sweat glands, and was associated with gland damage and regeneration. We conclude that C3 mediates Th1/Th17 polarization in human T-cell activation and skin GVHD in patients.