Cell-Based Quantification of Chronic Wasting Disease Prions

Cell-based measurement of prion infectivity is currently restricted to experimental strains of mouse-adapted scrapie. Having isolated cell cultures with susceptibility to prions from diseased elk, we describe a modification of the scrapie cell assay allowing evaluation of prions causing chronic wasting disease, a naturally occurring transmissible spongiform encephalopathy. We compare this cervid prion cell assay to bioassays in transgenic mice, the only other existing method for quantification, and show this assay to be a relatively economical and expedient alternative that will likely facilitate studies of this important prion disease.Prions consist largely or entirely of PrP^Sc, a β-sheet-rich conformer of the prion protein (PrP). During disease, PrP^Sc coerces the normal PrP^C protein to adopt the PrP^Sc conformation. While protease-sensitive forms of PrP^Sc exist (20), PrP^Sc is usually partially resistant to limited proteinase K (PK) digestion (4). Bioassays in susceptible animals have, until recently, been the sole means of assessing prion infectivity. The scrapie cell assay (SCA) (12), which relies on detection of protease resistant PrP^Sc, while a substantial advance, has been limited to the detection of mouse-adapted scrapie prions, and the development of analogous systems for naturally occurring prions is a high priority. Chronic wasting disease (CWD), a burgeoning epidemic of deer, elk, and moose, is of particular importance.We first generated a cell line susceptible to infection by cervid prions. While rabbit kidney epithelial RK13 cells expressing sheep, mouse, and bank vole PrP supported prion replication from the corresponding species (8, 14, 22), expression of human PrP did not confer susceptibility to human prions (14). We produced RK13 cells expressing elk PrP (RKE cells) and infected them with CWD brain homogenates (5). To analyze cervid PrP^Sc (CerPrP^Sc) by Western blotting, detergent extracts containing equal amounts protein were treated with 40 mg/ml PK for 1 h at 50°C and centrifuged for 1 h at 100,000 × g. Alternately, CerPrP^Sc in cells was analyzed by cell blotting (5). Infection of RKE cells with CWD resulted in detectable CerPrP^Sc 3 passages after infection; however, the progressive reduction of CerPrP^Sc upon repeated passage (Fig. ​(Fig.11 A) showed that infection was not sustained.Open in a separate windowFIG. 1.Characterization of cell cultures for studying CWD prions. (A) Western blots showing accumulation of CerPrP^Sc in RKE cells challenged with CWD brain homogenates from elk isolate 012-09442, passaged in Tg5037 mice (RKE-CWD) (left) and Elk21⁺ cells (right). Passage numbers (p) of cell cultures are indicated. (B) Expression of CerPrP^C and HIV-Gag and accumulation of CerPrP^Sc in RKE and RKE-Gag cells infected with CWD brain homogenates. Cultured cells were also analyzed by cell blotting (right). (C) Bioassay of Elk21⁺ cells propagating elk CWD 012-09442 prions (filled circles), elk CWD 012-09442 prions in Tg5037 mice (filled squares), uninfected RKE-Gag (open circles), Elk21⁻ 13 passages after DS-500 treatment (open triangles), Elk21⁻ 30 passages after DS-500 treatment (filled triangles), the Elk21-3 clone (open diamonds), and the Elk21-9 clone (open squares). (D) Western blots of CerPrP^C (100 and 50 μg total protein loaded in each case) and CerPrP^Sc (200, 100, and 50 μg total protein loaded in each case) produced in Elk21⁺ cells and Tg5037 mice inoculated with Elk21⁺ cell extracts. (E to H) CerPrP^Sc deposition in the hippocampus (E and G) and thalamus (F and H) of Tg5037 mice inoculated with either Elk21⁺ (E and F) or uninfected RKE-Gag (G and H) cell extracts. (I) Western blots demonstrating susceptibility of Elk21-3, Elk21-9, and Elk21⁻ to reinfection with elk CWD 012-09442 prions. For each cell line, the first two lanes show extracts from mock (phosphate-buffered saline [PBS])-infected cells, while the second two lanes show extracts from cells exposed to CWD brain homogenates. In all Western blots, samples were either PK treated (+) or untreated (−), and the positions of protein molecular mass markers at 37, 25, and 20 kDa (from top to bottom) are shown.Since previous reports demonstrated that retroviral Gag mediated enhanced release of mouse-adapted scrapie from cell cultures (15), RKE cells were further transfected with pcDNA3-gag expressing the HIV-1 GAG precursor protein (9), generating RKE-Gag cells. CerPrP^Sc levels in infected RKE-Gag were enhanced ∼2-fold (Fig. ​(Fig.1B).1B). Clones of infected RKE-Gag and RKE cells were derived by limited dilution. Of 40 clones isolated in each case, single RKE and RKE-Gag clones produced CerPrP^Sc. While CerPrP^Sc was not detected beyond passage 4 of cloned RKE cells (data not shown), Fig. ​Fig.1A1A shows that CerPrP^Sc production in the infected RKE-Gag clone, referred to as Elk21⁺, was sustained for 67 passages in culture, which equates to ∼223 cell doublings.Other approaches for producing cells chronically infected with CWD brain homogenates, including infection of N2a cells stably expressing elk PrP, were unsuccessful (Fig. ​(Fig.2),2), either because N2a cells are resistant to CWD brain homogenates or because CerPrP^C-to-CerPrP^Sc conversion is inhibited by expression of endogenous mouse PrP^C (21).Open in a separate windowFIG. 2.Lack of susceptibility of N2a cells expressing elk PrP to CWD. (A) Western blot showing elk and deer PrP^C expression in N2a cells. (B) Elk PrP^c-expressing N2a cells (N2a-ElkPrP) infected with CWD isolates remain uninfected after four passages. Pairs of lanes show extracts of N2a-ElkPrP cells challenged with brain homogenates from diseased Tg(CerPrP-E226)5037^+/− mice infected with elk isolates 012-022012, 012-09442, 99W12389, and 7178-47 (from left to right). Results are also shown for Elk21⁺ cells and CWD-infected Tg(CerPrP-E226)5037^+/− mice. Blots were probed with MAb 9E9, which recognizes only cervid PrP. Samples were either PK treated (+) or untreated (−), and the positions of protein molecular mass markers at 37, 25, and 20 kDa (from top to bottom) are shown.After 25 passages, Elk21⁺ cells were bioassayed in Tg(CerPrP-E226)5037^+/− mice expressing elk PrP (3), referred to as Tg5037 mice. Mice developed prion disease with a mean incubation time of 112 ± 1 days. Tg5037 mice inoculated with the same prions as those used to produce Elk21⁺ cells developed disease with a mean incubation time of 126 ± 2 days, while Tg5037 mice challenged with RKE-Gag cells remained asymptomatic (Fig. ​(Fig.1C).1C). Mice were inoculated with infected brain and cell culture preparations containing similar amounts of PrP^Sc as quantified by Western blot analysis. For bioassays of uninfected cells, mice were inoculated with preparations containing amounts of total protein equivalent to those in infected cell cultures.Levels of CerPrP^C and CerPrP^Sc and their electrophoretic migration and glycosylation patterns differed between Elk21⁺ cells and Tg5037 mice (Fig. ​(Fig.1D1D and ​and2).2). CerPrP^Sc deposition in the brains of Tg5037 mice infected with Elk21⁺ cells was diffuse and granular (Fig. 1E and F), in accordance with previous reports (3); no disease occurred in Tg5037 mice inoculated with RKE-Gag cells (Fig. 1G and H).Elk21⁺ cells were treated with the antiprion compound dextran sulfate 500 (DS-500) (7, 11, 13). CerPrP^Sc was undetectable after 5 weeks and did not reemerge when cells were returned to medium lacking drug (Fig. ​(Fig.1I).1I). Tg5037 mice remained asymptomatic for >355 days following inoculation (Fig. ​(Fig.1C).1C). Cells cured of PrP^Sc by DS-500, referred to as Elk21⁻ cells, retained the ability to sustain production of CerPrP^Sc when rechallenged with elk CWD brain homogenates (Fig. ​(Fig.1I).1I). The process of cloning of Elk21⁺ cells after 58 passages in culture also resulted in elimination of PrP^Sc in a subset of subclones. While Western and cell blotting detected CerPrP^Sc in 3 subclones, 11 subclones did not produce CerPrP^Sc. Of the 11 “negative” subclones rechallenged with CWD brain homogenates, 10 produced CerPrP^Sc (for example, clone Elk21-3 [Fig. ​[Fig.1I]),1I]), while clone Elk21-9 was resistant to CWD (Fig. ​(Fig.1I).1I). The CWD-free statuses of clones Elk21-3 and Elk21-9 were confirmed in Tg5037 mice (Fig. ​(Fig.1C1C).We adapted the SCA (12, 16, 17) to visualize infected Elk21⁻ or Elk21-3 cells. We refer to this as the cervid prion cell assay (CPCA) (see the supplemental material). Briefly, susceptible Elk21⁻ cells in 96-well plates were exposed to serial dilutions of CWD brain homogenates ranging from 10⁻² to 10⁻⁵ in a volume of 100 μl. Cell cultures were split once at 1:4 and twice at 1:7, which effectively diluted out CerPrP^Sc in the inoculum. Inclusion of RK13 cells stably transfected with empty vector (RKV cells) showed that positive spots detected after three splits were the result of newly generated CerPrP^Sc. After the final passage, 20,000 cells were filtered onto Multiscreen IP 96-well, 0.45-μm filter plates (enzyme-linked immunospot [ELISPOT] assay plates; Millipore, Billerica, MA) or AcroWell 96-well, 0.45-μm BioTrace filter plates (Pall, East Hills, NY). Cells were subjected to PK digestion and denaturation with guanidinium thiocyanate. CerPrP^Sc-producing cells were detected by an enzyme-linked immunosorbent assay (ELISA) using anti-PrP monoclonal antibody (MAb) 6H4, followed by alkaline phosphatase (AP)-conjugated secondary anti-mouse IgG, and developed with NBT/BCIP. Images were scanned with CTL ELISPOT equipment, and spot numbers were determined using ImmunoSpot3 software (Cellular Technology, Ltd., Shaker Heights, OH). Figure ​Figure33 A depicts magnifications of ELISPOT filters of infected Elk21⁻ cells.Open in a separate windowFIG. 3.Quantification of elk CWD prion infectivity by the transgenic mouse bioassay and the cervid prion cell assay. (A) Representative wells of an ELISPOT plate showing spots given by duplicate Elk21⁺ cells exposed to 3-fold serial dilutions of pooled elk CWD brain homogenates, between 10⁻³ and 10^−4.4. (B) Double-logarithmic plot of spot number versus brain homogenate dilution showing the linear response of the CPCA. Elk21⁻ cells infected with dilutions of pooled elk CWD brain homogenates (open circles) and pooled elk CWD brain homogenates passaged in Tg5037 mice (filled circles). In each case, the mean is derived from 6 independent experiments performed in triplicate, with error bars indicating the standard errors of the means (SEM). (C) Responsiveness of Elk21⁻ and Elk21-3 cells to various CWD brain homogenates. The cells were infected with serial 1:3 dilutions of homogenates of CWD-infected brains and subjected to the CPCA. In each case, the dilution required to yield 300 positive cells per 20,000 cells after the third split was calculated. Solid black line, CPCA of CWD brain homogenates from diseased elk brain, using Elk21⁻ cells; solid gray lines, CPCA of CWD brain homogenates from diseased Tg5037 mice, using Elk21⁻ cells; dashed gray line, CPCA using Elk21-3 cells. Filled triangles, D10 CWD isolate; open circles, pooled elk CWD brain homogenate; filled circles, brain homogenate of Tg5037 mice infected with pooled elk CWD; filled diamonds, 012-09442 CWD isolate; filled squares, 01-0306 CWD isolate.To determine the dose-response relationship of Elk21⁻ cells to CWD brain homogenates, we used a pooled elk CWD inoculum titrated in two different transgenic mouse lines (3, 6). In the case of Tg5037 mice, we estimated the titer to be 10^7.0 intracerebral (i.c.) 50% infective doses (ID₅₀)/g of brain, and the titer in Tg(CerPrP)1536^+/− mice expressing deer PrP was estimated at 10^7.2 i.c. ID₅₀/g (Table ​(Table1)1) (19).

TABLE 1.

CWD infectivity assays

Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer

While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.     

Chronic Wasting Disease (CWD) Susceptibility of Several North American Rodents That Are Sympatric with Cervid CWD Epidemics

Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted.Chronic wasting disease (CWD) is a contagious transmissible spongiform encephalopathy (TSE), or prion protein (PrP) disease, naturally transmitted among members of the deer family Cervidae (29). The putative infectious agent of prion diseases is a misfolded isoform of the PrP referred to as PrP^d that typically possesses unusual resistance to protease degradation. A hallmark of prion diseases is the accumulation of PrP^d in the brain, along with associated pathologies. CWD epidemics are occurring in free-ranging cervid populations at several locations in North American and involve several Cervidae species/subspecies (deer, Rocky Mountain elk, and moose [Odocoileus spp., Cervus elaphus nelsoni, and Alces alces, respectively]) (3, 29). In cervids, the potential for direct transmission via saliva has been demonstrated (13, 21), and indirect transmission via environmental contamination has been observed (22). Once in the environment, the agent remains infectious to cervids for extended periods (22). Secretions, excreta, and the tissues of dead animals are potential sources for environmental contamination; their relative importance is unknown. Other wildlife species are inevitably exposed to infectious material in the environment (25).To examine the potential for noncervid species to support CWD transmission, we intracerebrally challenged four species of native North American rodents: meadow voles (Microtus pennsylvanicus), red-backed voles (Myodes gapperi), white-footed mice (Peromyscus leucopus), and deer mice (P. maniculatus). These species all occur in locations undergoing cervid CWD epidemics (Fig. ​(Fig.1),1), and their opportunistic scavenging behaviors make exposure to infectious material highly likely.Open in a separate windowFIG. 1.Overlap of rodent distributions and CWD epidemics. Black dots show known locations of CWD in free-ranging cervids, and the green is the rodent''s distribution. (A) Meadow voles; (B) red-backed voles; (C) white-footed mice; (D) deer mice.We selected meadow voles because early studies by Chandler and Turfrey (6, 7) demonstrated efficient intracerebral transmission of scrapie to the field vole Microtus agrestis. Agrimi''s group (1, 10) reported similar findings for scrapie transmission into the European bank vole Myodes glareolus, motivating us to look at the North American red-backed vole, which some consider to be a semispecies with respect to bank voles (11). We included Peromyscus spp. because of their abundance and likelihood of coming into contact with infected cervid tissue in the field.     

Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP^d) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP^d detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP^d identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.     

Evaluating Spatial Overlap and Relatedness of White-tailed Deer in a Chronic Wasting Disease Management Zone

Wildlife disease transmission, at a local scale, can occur from interactions between infected and susceptible conspecifics or from a contaminated environment. Thus, the degree of spatial overlap and rate of contact among deer is likely to impact both direct and indirect transmission of infectious diseases such chronic wasting disease (CWD) or bovine tuberculosis. We identified a strong relationship between degree of spatial overlap (volume of intersection) and genetic relatedness for female white-tailed deer in Wisconsin’s area of highest CWD prevalence. We used volume of intersection as a surrogate for contact rates between deer and concluded that related deer are more likely to have contact, which may drive disease transmission dynamics. In addition, we found that age of deer influences overlap, with fawns exhibiting the highest degree of overlap with other deer. Our results further support the finding that female social groups have higher contact among related deer which can result in transmission of infectious diseases. We suggest that control of large social groups comprised of closely related deer may be an effective strategy in slowing the transmission of infectious pathogens, and CWD in particular.     

Infectious Prions in Pre-Clinical Deer and Transmission of Chronic Wasting Disease Solely by Environmental Exposure

Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.     

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrP^Sc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.     

Chronic Wasting Disease in Bank Voles: Characterisation of the Shortest Incubation Time Model for Prion Diseases

In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv^109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv^109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv^109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrP^Sc (PrP^res) in the same brain regions. Despite the low PrP^res levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 10^8,4 i.c. ID₅₀ infectious units per gram. Bv^109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv^109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv^109ICWD showed unique characteristics of “virulence”, low PrP^res accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrP^Sc, neurodegeneration and infectivity.     

Seroepidemiological Survey of Coxiella burnetii in Domestic Cats in Japan

Cats are assumed to be one of the most important reservoirs of causative agent of human Q fever especially in urban areas. There is no evidence of Coxiella burnetii infection in cats in Japan prior to this. Sera from 100 cats, collected in various parts of Japan, were examined for antibody against C. burnetii. Sixteen out of the 100 samples contained antibodies against C. burnetii. The prevalence of the antibody decreased from the northeastern to the southwestern part of Japan. A high prevalence of the antibodies was observed in sera from cats of more than four years of age. It is difficult to deny that cats would be one of the important sources of human Q fever in Japan.     

Empirical Estimation of R 0 for Unknown Transmission Functions: The Case of Chronic Wasting Disease in Alberta

We consider the problem of estimating the basic reproduction number R₀ from data on prevalence dynamics at the beginning of a disease outbreak. We derive discrete and continuous time models, some coefficients of which are to be fitted from data. We show that prevalence of the disease is sufficient to determine R₀. We apply this method to chronic wasting disease spread in Alberta determining a range of possible R₀ and their sensitivity to the probability of deer annual survival.     

Frequency-dependent Food Selection by Domestic Cats: A Comparative Study

Preferences for common food types (‘apostatic selection’) have been demonstrated in a wide variety of vertebrate predators, yet there are few examples of preferences for rare food types (‘anti-apostatic selection’). Anti-apostatic selection is predicted to occur when, among other things, there are nutritional benefits to be gained from the consumption of a mixed diet. We tested this hypothesis by examining the frequency-dependent food preferences of domestic cats (Felis silvestris catus) with different nutritional histories. Subjects were classified as being either nutritionally ‘experienced’ (farm and rescue shelter cats, with a history of scavenging for nutritionally variable foods) or nutritionally ‘inexperienced’ (cats reared indoors on high-quality, nutritionally complete diets). We tested for frequency dependence by allowing individuals and groups of cats from the two groups to select from high-density mixtures of two types of artificial food pellet. In experiments on individual cats, nutritionally experienced subjects showed significant anti-apostatic selection, whereas inexperienced cats produced only a weak anti-apostatic trend. In experiments on groups of cats, both inexperienced and experienced groups showed significant anti-apostatic selection. The apparent inconsistency between individual and group results could be explained in terms of the additional anti-apostatic effects that result from variation among individuals in group foraging situations (i.e. when the effects of individuals are pooled). Because other behavioural explanations, such as perceptual contrast and sampling effects, were unlikely to have influenced our results, we conclude that the differences in selection between experienced and inexperienced individuals were probably due to the differing extent to which the consumption of a mixed diet was beneficial. These experiments may offer some insight into the success of the domestic cat in urban areas: although obligate carnivores, they appear to possess flexible feeding strategies which will tend to allow them to select a reasonably balanced diet from nutritionally variable resources in, for example, refuse bins.     

Leukocyte Bactericidal Activity in Chronic Granulomatous Disease: Correlation of Bacterial Hydrogen Peroxide Production and Susceptibility to Intracellular Killing

Susceptibility of bacteria to intracellular killing by polymorphonuclear leukocytes from a patient with chronic granulomatous disease could be correlated with bacterial hydrogen peroxide production.     

Landscape-Level Variation in Disease Susceptibility Related to Shallow-Water Hypoxia

Diel-cycling hypoxia is widespread in shallow portions of estuaries and lagoons, especially in systems with high nutrient loads resulting from human activities. Far less is known about the effects of this form of hypoxia than deeper-water seasonal or persistent low dissolved oxygen. We examined field patterns of diel-cycling hypoxia and used field and laboratory experiments to test its effects on acquisition and progression of Perkinsus marinus infections in the eastern oyster, Crassostrea virginica, as well as on oyster growth and filtration. P. marinus infections cause the disease known as Dermo, have been responsible for declines in oyster populations, and have limited success of oyster restoration efforts. The severity of diel-cycling hypoxia varied among shallow monitored sites in Chesapeake Bay, and average daily minimum dissolved oxygen was positively correlated with average daily minimum pH. In both field and laboratory experiments, diel-cycling hypoxia increased acquisition and progression of infections, with stronger results found for younger (1-year-old) than older (2-3-year-old) oysters, and more pronounced effects on both infections and growth found in the field than in the laboratory. Filtration by oysters was reduced during brief periods of exposure to severe hypoxia. This should have reduced exposure to waterborne P. marinus, and contributed to the negative relationship found between hypoxia frequency and oyster growth. Negative effects of hypoxia on the host immune response is, therefore, the likely mechanism leading to elevated infections in oysters exposed to hypoxia relative to control treatments. Because there is considerable spatial variation in the frequency and severity of hypoxia, diel-cycling hypoxia may contribute to landscape-level spatial variation in disease dynamics within and among estuarine systems.     

Crohn's Disease and Early Exposure to Domestic Refrigeration

Background

Environmental risk factors playing a causative role in Crohn''s Disease (CD) remain largely unknown. Recently, it has been suggested that refrigerated food could be involved in disease development. We thus conducted a pilot case control study to explore the association of CD with the exposure to domestic refrigeration in childhood.

Methodology/Principal Findings

Using a standard questionnaire we interviewed 199 CD cases and 207 age-matched patients with irritable bowel syndrome (IBS) as controls. Cases and controls were followed by the same gastroenterologists of tertiary referral clinics in Tehran, Iran. The questionnaire focused on the date of the first acquisition of home refrigerator and freezer. Data were analysed by a multivariate logistic model. The current age was in average 34 years in CD cases and the percentage of females in the case and control groups were respectively 48.3% and 63.7%. Patients were exposed earlier than controls to the refrigerator (X2 = 9.9, df = 3, P = 0.04) and refrigerator exposure at birth was found to be a risk factor for CD (OR = 2.08 (95% CI: 1.01–4.29), P = 0.05). Comparable results were obtained looking for the exposure to freezer at home. Finally, among the other recorded items reflecting the hygiene and comfort at home, we also found personal television, car and washing machine associated with CD.

Conclusion

This study supports the opinion that CD is associated with exposure to domestic refrigeration, among other household factors, during childhood.