Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.     

Neutering Dogs: Effects on Joint Disorders and Cancers in Golden Retrievers

In contrast to European countries, the overwhelming majority of dogs in the U.S. are neutered (including spaying), usually done before one year of age. Given the importance of gonadal hormones in growth and development, this cultural contrast invites an analysis of the multiple organ systems that may be adversely affected by neutering. Using a single breed-specific dataset, the objective was to examine the variables of gender and age at the time of neutering versus leaving dogs gonadally intact, on all diseases occurring with sufficient frequency for statistical analyses. Given its popularity and vulnerability to various cancers and joint disorders, the Golden Retriever was chosen for this study. Veterinary hospital records of 759 client-owned, intact and neutered female and male dogs, 1–8 years old, were examined for diagnoses of hip dysplasia (HD), cranial cruciate ligament tear (CCL), lymphosarcoma (LSA), hemangiosarcoma (HSA), and mast cell tumor (MCT). Patients were classified as intact, or neutered early (<12 mo) or late (≥12 mo). Statistical analyses involved survival analyses and incidence rate comparisons. Outcomes at the 5 percent level of significance are reported. Of early-neutered males, 10 percent were diagnosed with HD, double the occurrence in intact males. There were no cases of CCL diagnosed in intact males or females, but in early-neutered males and females the occurrences were 5 percent and 8 percent, respectively. Almost 10 percent of early-neutered males were diagnosed with LSA, 3 times more than intact males. The percentage of HSA cases in late-neutered females (about 8 percent) was 4 times more than intact and early-neutered females. There were no cases of MCT in intact females, but the occurrence was nearly 6 percent in late-neutered females. The results have health implications for Golden Retriever companion and service dogs, and for oncologists using dogs as models of cancers that occur in humans.     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.     

Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cells

Laminin-binding integrins form a complex with CD151, a member of the tetraspanin family suggested to be involved in the regulation of cell migration. In the epidermis, CD151 is localized with alpha3beta1 and alpha6beta4 integrins at cell-cell and cell-matrix contacts, respectively, characteristic structures of non-migrating cells. Taking advantage of a monoclonal antibody against CD151, TS151r, which epitope overlaps with the tetraspanin integrin-binding site, we have investigated the role of CD151 in epithelial cell migration. Under standard culture conditions, the migratory capacity of epithelial HaCaT cells on laminins is low, apparently due to endogenous laminin 5. However, challenging HaCaT cells with TS151r allows a re-arrangement of the actin cytoskeleton, dismantling of cell-cell and beta4 integrin-mediated cell-matrix contacts and cell migration. In vivo, free CD151 is absent in resting epithelial cells of interfollicular epidermis, and all CD151 is bound to integrins in intercellular and cell-matrix contacts. By contrast, free CD151 is present at intercellular contacts in the epithelial sheet lining the deeper region of anagen hair follicles, which is considered to contain migrating cells. Together, these results strongly suggest that dissociation of the CD151-integrin complex permits remodeling of epithelial cell interactions with the extracellular matrix and cell migration.     

Intake and digestion of horned guan Oreophasis derbianus diets measured in three Mexican zoos

We conducted nutritional analyses of diets offered to and ingested by seven pairs of horned guans (Oreophasis derbianus) in three zoos. Digestibility was calculated with individually housed birds (n=1 at each zoo). Diets offered varied widely among institutions, both in ingredients fed as well as in nutrient composition. Feeding selectivity was evident through differences in composition of diets offered vs. consumed, with fruit (bananas, grape, and/or plantain) and avocado (when offered) highly preferred; green leaves, poultry pellets, and other vegetables comprised lesser proportions of the diet. All facilities fed 2–3X more food than consumed, allowing a great degree of choice of preferred items and potentially consumption of nutritionally imbalanced diets—in particular, mineral constituents. Diets were highly digestible; dry matter (DM) digestion coefficients ranged from 70 to ∼90%; protein digestibility varied from 30 to 80%; fat was >90% digestible. Diet composition was compared with known nutritional requirements of domestic avian species, and feeding recommendations discussed. Despite the wide variability in nutrient composition of diets eaten (i.e. protein 6–10% of DM; fat 2–17% of DM), no overt health problems were noted and all pairs had successfully reproduced on these diets. It is suggested that horned guans may have nutrient requirements more similar to those suggested for other frugivorous birds than values determined for poultry as the physiologic model. Comparisons with native food items, as well as more detailed nutrient balance studies, may provide even better guidelines for captive management of this highly endangered species. Zoo Biol 28:319–330, 2009. © 2009 Wiley-Liss, Inc.     

Effects of Hurricanes on Rare Plant Demography in Fire‐Controlled Ecosystems

Hurricanes have dramatic effects on forest vegetation, but their effects on shrublands have rarely been studied. We analyzed the effects of three 2004 hurricanes—among the strongest on record in Florida—on vital rates of 12 rare plant species of pyrogenic interior Florida scrub and sandhill. Tree damage varied by vegetation type (being highest in areas with Pinus clausa) and was associated with debris deposition. Most rare species were minimally impacted by hurricanes. The two most frequently damaged species were the shrubs Prunus geniculata (11% of individuals) and Asimina obovata (7%); both were resilient to damage. Prunus geniculata had little mortality during the hurricane year but damaged plants had a temporary (1‐yr) reduction in relative growth rate. Prunus geniculata flowering was unaffected by hurricane damage. Hurricane damage had no effects on vital rates of A. obovata, Eriogonum longifolium var. gnaphalifolium, or Chrysopsis highlandsensis. Other species suffered little or no observable hurricane damage. Of 12 species analyzed, nine had similar annual survival in hurricane and nonhurricane years. Relatively low survival in the hurricane year (compared with other years) was linked to prehurricane drought or prescribed fire in two of three species. Thus, the 2004 hurricanes did not have important effects on populations of interior Florida scrub and sandhill plants, especially herbaceous species. This is in marked contrast to dramatic demographic responses to fire in central Florida and strong effects of hurricanes in coastal Florida, highlighting that these different disturbances may have divergent effects on vegetation and populations over short distances.     

Why on earth?: Evaluating hypotheses about the physiological functions of human geophagy

Geophagy has been hypothesized to be an adaptive behavior, either as a means to allay nutrient deficiency or to protect against ingested pathogens and toxins. Others have proposed that geophagy is non-adaptive, occurring either to allay hunger or as an epiphenomenon of nutrient deficiencies. This paper evaluates these hypotheses using 482 published cultural-level accounts of human geophagy and 330 accounts of geophagy among 297 species of mammals, birds, and reptiles. Information was extracted from reports of human geophagy to permit statistical analysis; reports of non-human geophagy were tabulated. Human geophagy did not parallel changes in nutrient requirements, occurred most frequently among children and pregnant women and in tropical areas (where pathogen densities are highest), and was associated with ingestion of toxic substances and gastrointestinal distress. Earth ingested by humans was craved and carefully selected and prepared; it had high clay content, but few bioavailable mineral nutrients. In primates, geophagy was associated with both protection from toxins and obtaining nutrients, whereas in other vertebrates it was associated mainly with obtaining nutrients. Our results indicate that human geophagy is best explained as providing protection from dietary chemicals, parasites, and pathogens, whereas animal geophagy may involve both micronutrient acquisition and protection.     

Presence of Human Papillomavirus DNA in Malignant Neoplasia and Non-Malignant Breast Disease

Breast cancer is the leading cause of cancer death among women worldwide. Multiple extrinsic and intrinsic factors are associated with this disease’s development. Various research groups worldwide have reported the presence of human papillomavirus (HPV) DNA in samples of malignant breast tumors. Although its role in mammary carcinogenesis is not fully understood, it is known that the HPV genome, once inserted into host cells, has oncogenic capabilities. The present study aimed to detect the presence of HPV DNA in 116 breast tissue biopsies and classify them according to their histology. It was found that 50.9% of the breast biopsies analyzed were malignant neoplasms, of which 74.6% were histologically classified as infiltrating ductal carcinoma. In biopsies with non-malignant breast disease, fibroadenoma was the most common benign neoplasm (39.1%). Detection of HPV DNA was performed through nested PCR using the external primer MY09/11 and the internal primer GP5+/6+. A hybridization assay genotyped HPV. HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% non-malignant breast disease (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. However, there are no statistically significant differences between these groups and the existence of HPV DNA (p = 0.2521). Its presence was more frequent in non-malignant alterations than in malignant neoplasias. The most frequent genotypes in the HPV-positive samples were low-risk (LR) HPV-42 followed by high-risk (HR) HPV-31.