Energetic costs of mange in wolves estimated from infrared thermography.

Parasites, by definition, extract energy from their hosts and thus affect trophic and food web dynamics even when the parasite may have limited effects on host population size. We studied the energetic costs of mange (Sarcoptes scabiei) in wolves (Canis lupus) using thermal cameras to estimate heat losses associated with compromised insulation during the winter. We combined the field data of known, naturally infected wolves with a data set on captive wolves with shaved patches of fur as a positive control to simulate mange-induced hair loss. We predict that during the winter in Montana, more severe mange infection increases heat loss by around 5.2-12 MJ per night (1,240-2,850 kcal, or a 65-78% increase) for small and large wolves, respectively, accounting for wind effects. To maintain body temperature would require a significant proportion of a healthy wolf's total daily energy demands (18-22 MJ/day). We also predict how these thermal costs may increase in colder climates by comparing our predictions in Bozeman, Montana to those from a place with lower ambient temperatures (Fairbanks, Alaska). Contrary to our expectations, the 14°C differential between these regions was not as important as the potential differences in wind speed. These large increases in energetic demands can be mitigated by either increasing consumption rates or decreasing other energy demands. Data from GPS-collared wolves indicated that healthy wolves move, on average, 17 km per day, which was reduced by 1.5, 1.8, and 6.5 km for light, medium, and severe hair loss. In addition, the wolf with the most hair loss was less active at night and more active during the day, which is the converse of the movement patterns of healthy wolves. At the individual level, mange infections create significant energy demands and altered behavioral patterns, this may have cascading effects on prey consumption rates, food web dynamics, predator-prey interactions, and scavenger communities.

Prioritizing genomic applications for action by level of evidence: a horizon-scanning method.

As evidence accumulates on the use of genomic tests and other health-related applications of genomic technologies, decision makers may increasingly seek support in identifying which applications have sufficiently robust evidence to suggest they might be considered for action. As an interim working process to provide such support, we developed a horizon-scanning method that assigns genomic applications to tiers defined by availability of synthesized evidence. We illustrate an application of the method to pharmacogenomics tests.

Ultrastructure of Gonionchus australis (Xyalidae, Nematoda).

Observations are reported on the ultrastructure of the buccal cavity, body cuticle, spermatids, spermatozoa, male genitalia, and caudal glands of Gonionchus australis. The buccal cuticle is a continuation of the pharyngeal cuticle. Anteriorly it is secreted by arcade tissue and overlaps the mouth rim; laterally it forms longitudinal tooth ridges. The non-annulated cephalic cuticle differs sharply from the remainder of the body wall cuticle. The cortical and basal zones become much thinner, while a largely structureless, lucent median zone expands to fill the bulk of the lips and lip flaps. Spermatids possess fibrous bodies, multimembrane organelles, mitochondria, and compact chromatin. The spermatozoa of G. australis resemble those of most other nematodes by the absence of the nuclear envelope and presence of fibrous bodies, mitochondria, and compact chromafin. The ejaculatory duct possesses microvilli. Two ejaculatory glands lie beside the duct. Two neurons are located within each spicule and each part of the paired gubernaculum. Caudal gland nuclei are large, with dispersed chromatin. The ducts of all three caudal glands are filled with secretory vesicles.

Intratype variation in 12 human papillomavirus types: a worldwide perspective.

In this study, we have examined intratype human papillomavirus (HPV) sequence variation in a worldwide collection of cervical specimens. Twelve different HPV types including HPV-18, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-58, HPV-59, HPV-68 (ME180), MM9/PAP238A (recently designated HPV-73), and a novel partial genomic HPV sequence designated MM4/Wl3B were analyzed in this study. Cervical specimens were collected as part of epidemiological investigations conducted in New Mexico and an international study of invasive cervical cancer (IBSCC). Specimens from several countries including Argentina, Brazil, Bolivia, Benin, Cuba, Colombia, Chile, Germany, Mali, Panama, Paraguay, Spain, Algeria, Uganda, Guinea, Tanzania, Indonesia, Philippines, Thailand, and the United States were evaluated. Specimen DNAs were subjected to amplification with the MY09/11 L1 consensus PCR system. The PCR products were cloned, and an approximately 410-bp region in the L1 open reading frame was sequenced from 146 specimens (approximately 60,000 bp). Within a single HPV type, nucleotide diversity varied between 0.2 and 2.9% (i.e., between any pair of variants) and the majority of nucleotide changes were synonymous (amino acid conserving). These data provide information pertinent to HPV diagnostic probe development and are potentially relevant to future rational vaccine strategies. Similarly, amino acid diversity varied between 0 and 5.1%. Some of these amino acid changes may represent markers of intertype evolutionary relationships. Presuming that HPVs have evolved under the same constraints as their corresponding hosts, the limited genetic diversity observed for all HPVs studied to date may reflect an evolutionary bottleneck occurring in both virus and host populations.

Human papillomavirus type 16 variant lineages in United States populations characterized by nucleotide sequence analysis of the E6, L2, and L1 coding segments.

Human papillomavirus type 16 (HPV16) nucleotide sequence variations in the E6 (nucleotide positions [nt] 104 to 559), L2 (nt 4272 to 5657), and L1 (nt 5665 to 7148) open reading frames (ORFs), and the long control region (nt 7479 to 7842), were examined in 29 selected United States isolates. Of 3,690 nucleotide positions, 129 (3.5%) varied. The maximum pairwise distance was 66 nucleotide differences, or 1.8%. Nucleotide variations within different genome segments were phylogenetically compatible, and nucleotide changes within E6, L2, and L1 contained phylogenetic information beyond that provided in the long control region. Most isolates were classified as members of HPV16 lineages that have been described previously. However, two novel phylogenetic branches were identified. The L2 ORF was the most variable coding segment. L2 synonymous and nonsynonymous nucleotide changes were distributed asymmetrically. The amino-terminal half of the L2 protein was remarkably conserved among all isolates, suggesting that the region is under evolutionary constraint. The amino-terminal region of the E6 ORF was relatively varied, especially at E6 amino acid positions 10 and 14. Several amino acid difference in the L1 ORF were observed between lineages. Forty-nine amino acid variations across all sequenced coding regions were observed. These amino acid differences may be relevant to differences in the generation of humoral or cell-mediated immune responses to HPV16 variants. Our data form a basis for considering HPV16 sequence variation in the rational design of vaccine strategies and as an epidemiologic correlate of cervical cancer risk.

Generation of entire human papillomavirus genomes by long PCR: frequency of errors produced during amplification.

Recently, several improvements of traditional PCR techniques have facilitated the amplification of significantly longer DNA target sequences. Here we report an improved method for amplification of entire human papillomavirus (HPV) genomes. Using rTth DNA polymerase, XL (Perkin-Elmer, Foster City CA), and the accompanying XL PCR buffer system, we have successfully amplified 8-kb genomes from approximately 10 copies of input reference strain HPV16 DNA. This long PCR (LPCR) method was subsequently used to amplify the entire HPV16 genome from clinical specimens. The fidelity with which the rTth DNA polymerase XL amplified target sequences under our chosen amplification conditions was estimated by partial sequencing of cloned LPCR products generated from cloned reference strain HPV16 genomes. A region spanning the HPV16 E6, E7, and part of the E1 open reading frames (ORFs) was sequenced in 29 clones. A total of 33 nucleotide substitutions were observed in the 23.5 kb sequenced. This corresponds to an error frequency of approximately one error per 700 bases. Finally, LPCR methods were used to amplify entire, novel HPV genomes from clinical specimens. LPCR primer pairs were designed for amplification of seven potentially novel HPV types. Amplicons of approximately 8 kb were generated from five of the seven HPV types targeted. One of the LPCR-generated novel genomes, CP141, was subsequently cloned and a partial sequence was determined.

Hypnotherapy as a treatment for atopic dermatitis in adults and children.

Eighteen adults with extensive atopic dermatitis, resistant to conventional treatment, were treated by hypnotherapy, with statistically significant benefit (P < 0.01) measured both subjectively and objectively, which was maintained at up to 2 years where results were available. Twenty children with severe, resistant atopic dermatitis were treated by hypnosis. All but one showed immediate improvement, which was maintained at the following two clinic appointments. In 12 children, replies to a questionnaire at up to 18 months after treatment, showed that 10 had maintained improvement in itching and scratching, nine in sleep disturbance, and seven maintained improvement in itching and scratching, nine in sleep disturbance, and seven maintained improvement in mood.

The genomes of the animal papillomaviruses European elk papillomavirus, deer papillomavirus, and reindeer papillomavirus contain a novel transforming gene (E9) near the early polyadenylation site.

We report that the genomes of reindeer papillomavirus (RPV), European elk papillomavirus (EEPV), and deer papillomavirus (DPV) contain a short conserved translational open reading frame (ORF), E9, which is located between the E5 ORF and the early polyadenylation site. In RPV, DPV, and EEPV, E9 ORFs have the potential to encode extremely hydrophobic polypeptides of approximately 40 amino acids. In mouse C127 cells transformed by EEPV and RPV, there exists a unique, abundant mRNA species of approximately 700 nucleotides which has the capacity to encode an E9 polypeptide. This mRNA is transcribed from a previously unrecognized promoter at position 4030 in the EEPV genome. The EEPV E9 ORF exhibits weak transforming activity in C127 cells and primary rat embryo fibroblasts. We also show that EEPV E5 is the major oncogene in the EEPV genome when assayed in C127 cells, although it is less efficient in transformation than the E5 genes of bovine papillomavirus type 1, DPV, and RPV.

A novel human papillomavirus sequence from an international cervical cancer study.

Novel human papillomaviruses (HPVs) were sought as part of a recent international study of > 1000 invasive cervical carcinomas. A single novel HPV designated IS39 was identified in this study. IS39 is most closely related to another novel virus, W13B (MM4), variants of W13B (MM4), and HPV-51.

Anomalous transforming behavior of a bovine papillomavirus type 1 mutant with an upstream promoter mutation.

We show that the previously described high-transformation phenotype of the bovine papillomavirus type 1 mutant BPV730 is manifested only in an E6-dependent cell transformation assay. The BPV730 mutation was associated with superinduction of the putative E6 promoter, P89, after cycloheximide treatment, and with reduced activity of the P3080 promoter.

Segregation properties of bovine papillomaviral plasmid DNA.

Essentially complete segregation of replication-competent BPV-1 plasmid DNA species was observed in daughter subclones derived from primary co-transformed C127 cell lines. Thus, whereas primary co-transformants retained both of two distinguishable co-transfected plasmid species, subcloning experiments revealed that morphologically transformed daughter subclones derived from such co-transformed cell lines contained only one species of viral plasmid DNA. Similar results were obtained with each of two conveniently marked replication and transformation-competent mutants: one with a linker-insertion in the viral upstream regulatory region, and one with a 260 base-pair deletion within the L2 (late) gene, which has no recognized role in plasmid replication or stability. Morphological revertant cell clones that contained no detectable viral plasmid DNA genomes were also isolated at a surprisingly high frequency from clonal wild-type BPV-1 transformed cell lines and from cell lines transformed by various BPV-1 mutants. Further co-transfection experiments were done with a combination of transformation-competent and transformation-defective BPV-1 genomes to investigate a possible role for a viral oncogene in plasmid persistence. In this case, elimination of the transformation-defective mutant was observed after the initial establishment of both input genomes as replicating plasmids in cell clones morphologically transformed by the transformation-competent viral mutant with an intact E5 oncogene. No cell subclones were isolated that contained only the transformation-defective mutant, implying that it was defective in long-term plasmid persistence. Our results indicate that there is significant randomization in the processes of replication and/or partitioning of the BPV-1 genome in mouse C127 cells, and, in combination with previous observations, also suggest that BPV plasmid persistence in C127 cell lines may be the result of a selective proliferative advantage conferred on virus-infected cells by viral oncogene-induced cell growth transformation.

Fine Structure of the Head and Cervical Region of Ceramonema carinatum (Chromadorida: Ceramonematidae).

Structure of the head and cervical region of Ceramonema carinatum (Chromadorida: Ceramonematidae) was described from transmission electron microscopy of serial transverse and longitudinal sections of two females. An unbroken massive cortical layer encompasses the head, except where three thin liplets surround the mouth. A large flask-shaped buccal cavity, with simpler less dense cuticle identical with that of the pharynx, abuts the body cuticle just within the mouth. Myoepithelial ceils constituting the buccal and pharyngeal regions were described. Sixteen head sensilla, the amphids, and dorsal and ventral internal sensilla were identified and described, each with associated sheath and socket cells. Ultrasturcture of the head was compared with that of other nematodes. Arrangement of sensilla resembled that of Monhysterida and Rhabditida with some significant variations, such as prominent longitudinally arranged intracellular organelles containing many microtubules associated with the amphids. The buccal cavity was almost entirely pharyngeal in character. A well-developed system of structural fibrils and abundant hemidesmosomes were notable features.

Structure of the Cuticle of Ceramonema carinatum (Chromadorida: Ceramonenatidae).

The cuticle of Ceramonema carinatum (Chromadorida: Ceramonematidae) is described and illustrated from scanning and transmission electron microscopy. Each of ca. 200 annules is composed of a single ring with eight external flat faces (plates), which are divided by longitudinal ridges formed by pairs of parallel upstanding vanes. Vanes and plates overlap those of the adjacent annules. Longitudinal ridges extend from the cephalic capsule to the tail spike. On the cephalic capsule a simple ridge extends each of the eight ridges to a position just anterior to the amphid. Cuticular plates are formed from the electron-dense cortical layer and contain lacunae filled with fine fibrils. The vanes are denser, with laminations on a central core. In the annular grooves between the plates there is an electron-lucent layer, which it is suggested, by comparison with other nematodes, is the basal layer. An epicuticle overlies the cortical plates, the vanes, and the interannular lucent layer. Cuficular structure is compared with that of other Ceramonematidae and related nematodes.

Structure of the Cuticle of Metadasynemoides cristatus (Chromadorida: Ceramonematidae).

Structure of the cuticle of Metadasynemoides cristatus (Chromadorida: Ceramonematidae) is examined by light, scanning, and transmission electron microscopy. The nematode has more than 600 annuli, and each annulus has eight cuticular plates. Eight longitudinal ridges, beginning on the cephalic capsule, extend the whole length of the body. Where a ridge traverses an annulus, it forms a complicated articulating structure of overlapping vanes. Within the electron-dense cortical layer, from which the cuticular plates are formed, there are spaces crossed by fine fibrillae, forming what have been termed "vacuoles" by light microscopists. There is an epicuticle and a continuous lucent basal layer. There appears to be no median layer. The cuticle lining of the esophagus and that forming the circum-oral ridge is of much simpler construction.

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences.

A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the "thylakoid-transfer domain" of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.

Generation and isolation of cyanobacterial inside-out thylakoid vesicles.

A method has been designed to prepare inside-out thylakoid vesicles from a cyanobacterial species (Phormidium laminosum). Everted thylakoid vesicles could be generated by Yeda press treatment after induced membrane pairing. Membrane pairing was induced either by addition of high concentrations of Mg2+ ions or by lowering the pH of the fragmentation media. The inside-out vesicles were isolated by aqueous polymer two-phase partition. The membrane orientation was determined by proton translocation studies and freeze-fracture electron microscopy.

The measurement of ethylenethiourea and ethyleneurea in the rat and common marmoset Callithrix jacchus after zineb (zinc ethylenebisdithiocarbamate) dosing.

The metabolism of the fungicide zineb, (zinc ethylenebisdithiocarbamate), has been studied in the rat and the marmoset. 2. It was found in both species that a relatively large proportion (21-22%) of the original zineb administered was detectable in the excreta as ethylenethiourea (ETU) a known mutagen, teratogen and carcinogen. 3. A further proportion (2-5%) was determined to be ethyleneurea which is a metabolite of ETU. 4. Results of comparative experiments in marmosets revealed that ETU was photolabile in the presence of excreta, thus showing the importance of conducting the studies in the dark.

Ocular Toxocara canis infections: diagnosis by enzyme immunoassay.

An enzyme-linked immunoassay (ELISA) was used to establish the sero-prevalence of Toxocara canis infection in several New Zealand populations including those who, through environment or occupation, had close contact with dogs. The prevalence of positive ELISA values found reflected the degree of dog contact, e.g. positive ELISA's occurred in 2.8% of urban adults, 13.9% of dog breeders and 28.4% of hydatid control officers emphasizing that a history of dog contact needs to be considered when ELISA results are interpreted in a clinical context. Ophthalmoscopy of 102 hydatid officers, 28.4% of whom were seropositive, disclosed no evidence of ocular toxocariasis and suggested that the officers might be immunoprotected. Preliminary immunoblot analysis of the range of toxocara excretory/secretory antigens bound to serum immunoglobulin from patients with presumed visceral larva migrans and ocular toxocariasis, showed the same reactivity profile as a positive reference serum and serum from a seroresponsive hydatid control officer.

The relationship between toxocara ELISA absorbance values and toxocara ELISA titres: a comparative study.

Enzyme-linked immunosorbent assays (ELISAs) may be made by a variety of techniques at one or several serum dilutions which is very confusing for clinicians. This study compares the ELISA results from two centres on sera from 12 patients investigated for ocular toxocariasis by two common ELISA methods--the absorbance and titration techniques. With one exception, ELISA results by the two methods correlated well. For reasons outlined, the absorbance ELISA method is likely to remain the preferred method in New Zealand and Australia. Attention is drawn to the implications of the serum response to Toxocara canis larvae, and the need to evaluate ELISA results against clinical findings is emphasized.

Toxocariasis: a serological survey of blood donors in the Australian Capital Territory together with observations on the risks of infection.

The sera from 660 healthy blood donors from Canberra were tested for antibodies to Toxocara canis by the ELISA test. The results were compared with those from patients with suspected or confirmed visceral larva migrans or ocular toxocariasis. Over 7% of Canberra sera showed elevated levels of antibody reacting with T. canis antigen. Sera from patients resident in Australia with other helminth parasites did not cross-react with T. canis antigen in our tests. However, studies of sera collected in several tropical countries with other parasitic infection, show that cross-reactions with other parasites are possible. The use of purified glycoprotein antigen does not alter the possibility of cross-reaction. Observations and experiments show that people in Canberra may be exposed to the infective eggs of T. canis.