Host-genotype and agent effects in scrapie incubation: Change in allelic interaction with different strains of agent

Summary Incubation period in mice of ME7 scrapie agent is known to be controlled by the gene sinc. Evidence is presented that the same gene probably also determines incubation period for the 22A scrapie agent which has very different biological properties. Not only is there a reversal of the direction of gene effect when these agents are compared in mice homozygous for two different alleles of sinc, but there is also a different type of allelic interaction—absence of dominance with ME7 and overdominance with 22A. Other agents are mentioned which extend this range of allelic interaction even further. It is suggested that replication of scrapie agent depends on a multimeric host site and that the two alleles of sinc contribute different types of subunits to these sites.     

Absence of Eclipse Phase in Scrapie Mice

Field, Joyce and Keith¹ have claimed that the scrapie agent shows a viral characteristic by having an eclipse phase after being inoculated into the intracerebral region of mice. Three experiments, studying the accumulation of scrapie agent in spleen after intracerebral and intraperitoneal inoculation of mice, have not verified the conclusions of Field et al. A typical curve for the progression of scrapie activity after intracerebral inoculation (Fig. 1) shows no eclipse phase, nor was any observed in an experiment where the titre was examined every 2 days for the first 14 days after inoculation. Moreover, Field and his colleagues have not referred to similar studies with different results from theirs. Four independent groups of workers^2–6 have now examined the levels of scrapie activity in brain and spleen during the early stages after infection by three different routes and in none of these studies was there any clear indication of an eclipse phase in scrapie.     

Genetic control of scrapie and Creutzfeldt-Jakob disease in mice

Genetic control of experimental scrapie and Creutzfeldt-Jakob disease (CJD) was studied in inbred strains of mice by measuring the times from intracerebral inoculation with the agents to the onset of neurological dysfunction. Every strain of mice examined was susceptible to infection; however, a wide range of incubation times was found for both scrapie and CJD. New Zealand (NZ) mice, which eventually develop an autoimmune disorder, were inoculated intracerebrally with 10(6) ID50 units of the scrapie agent in a Chandler isolate. NZW mice showed incubation periods of less than 95 days; this is the shortest period recorded for any murine host with scrapie. In NZB and NZB X W F1 mice, the incubation periods were approximately 130 days and were similar to those in BALB/c and C57BL mice. Male and female NZ mice exhibited scrapie incubation periods of the same length. Similar results were obtained when B10.Q and C57BL/6J mice were inoculated intracerebrally with 10(4) ID50 units of the CJD agent in a K.Fu. isolate. These observations define a genetic locus or loci controlling the length of scrapie and CJD incubation periods; alleles coding for longer incubation times appear to be autosomal dominant. When congenic mice with a C57BL/10J background differing only in their H-2 haplotypes were studied, the results showed that the D subregion of the H-2 complex played a central role in controlling the length of the CJD incubation period. The q allele at the D subregion resulted in shorter incubation times, whereas the d allele resulted in long incubation times. The p, s, b, and k alleles gave intermediate incubation times. We propose the symbol PID-1 for designating this genetic locus which is located within the D subregion of the major histocompatibility (H-2) complex on murine chromosome 17. In addition, observations on congenic mice provide evidence for the influence of sex on CJD incubation periods. In some strains of inbred mice, males showed significantly shorter incubation periods compared with those for females with experimental CJD. These studies with inbred mice have defined previously unrecognized genes that control the length of scrapie and CJD incubation periods.     

Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility

The Tg(OvPrP4) mouse line, expressing the sheep prion protein, is a sensitive model crucial for the identification of the bovine spongiform encephalopathy agent possibly present in natural sheep spongiform encephalopathies. It was also previously demonstrated as susceptible to infection with natural scrapie isolates from sheep harbouring various genotypes. The performance of this new transgenic mouse line in scrapie strain characterization was further assessed by intracranial inoculation of five groups of Tg(OvPrP4) mice with brain homogenate of the wild type mouse-adapted scrapie strains, C506M3, 22A, 79A, 87V, or Chandler. The Tg(OvPrP4) mice were susceptible to the scrapie agent transmitted using mouse-adapted scrapie strains but not equivalently. Strains 87V and Chandler were most readily transmissible followed by 79A and C506M3. Strain 22A was the least transmissible. Clinical signs, survival data, spongiosis, and PrP^sc distribution were also reported. These various data demonstrate the possibility of distinguishing between scrapie strains. Our findings are discussed with regard to agent strain and host factors and already demonstrate the dissimilar susceptibilities of Tg(OvPrP4) mice to the different murine strains studied, thus, reinforcing their potential use in strain typing studies.     

Molecular and transmission characteristics of primary-passaged ovine scrapie isolates in conventional and ovine PrP transgenic mice

A more complete assessment of ovine prion strain diversity will be achieved by complementing biological strain typing in conventional and ovine PrP transgenic mice with a biochemical analysis of the resultant PrPSc. This will provide a correlation between ovine prion strain phenotype and the molecular nature of different PrP conformers associated with particular prion strains. Here, we have compared the molecular and transmission characteristics of ovine ARQ/ARQ and VRQ/VRQ scrapie isolates following primary passage in tg338 (VRQ) and tg59 (ARQ) ovine PrP transgenic mice and the conventional mouse lines C57BL/6 (Prnp^a), RIII (Prnp^a), and VM (Prnp^b). Our data show that these different genotypes of scrapie isolates display similar incubation periods of >350 days in conventional and tg59 mice. Facilitated transmission of sheep scrapie isolates occurred in tg338 mice, with incubation times reduced to 64 days for VRQ/VRQ inocula and to ≤210 days for ARQ/ARQ samples. Distinct genotype-specific lesion profiles were seen in the brains of conventional and tg59 mice with prion disease, which was accompanied by the accumulation of more conformationally stable PrPSc, following inoculation with ARQ/ARQ compared to VRQ/VRQ scrapie isolates. In contrast, the lesion profiles, quantities, and stability of PrPSc induced by the same inocula in tg338 mice were more similar than in the other mouse lines. Our data show that primary transmission of different genotypes of ovine prions is associated with the formation of different conformers of PrPSc with distinct molecular properties and provide the basis of a molecular approach to identify the true diversity of ovine prion strains.     

Unchanged survival rates of Shadoo knockout mice after infection with mouse-adapted scrapie

Previous studies have demonstrated that Shadoo (Sho), a GPI-linked glycoprotein encoded by the Sprn gene with a membrane localization similar to PrP^C, is reduced in the brains of rodents with terminal prion disease. To determine the functional significance of Sho in prion disease pathogenesis, Sho-deficient mice were generated by gene targeting. Sho knockout and control wild-type (WT) mice were infected with themouse-adapted scrapie strains 22L or RML. No significant differences in survival, the incubation period of prion disease or other disease features were observed between Sho mutant and WT mice. In this model of prion disease, Sho removal had no effect on disease pathogenesis.     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.     

Inhibition of Mitosis in Human Triploid Cells

TRIPLOIDY—the possession of three instead of two sets of chromosomes per cell-is a substantial cause of death in man. At least 1% of conceptions produce triploid zygotes, most of which are aborted by the end of the third month of pregnancy¹. Only a few triploid infants are reported to have survived birth but the reason is not known. Death in utero also seems to be the fate of triploid rats, mice and rabbits², but in amphibia³, Drosophila⁴ and many plants^5,6, triploid individuals are not only viable but do not differ markedly from normal diploids.     

Inflammation in Lafora Disease: Evolution with Disease Progression in Laforin and Malin Knock-out Mouse Models

Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a ^?/? (laforin knock-out) and Epm2b ^?/? (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 messenger RNAs (mRNAs) are significantly increased in Epm2a ^?/? mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα, and Il10ra mRNAs are significantly upregulated in Epm2b ^?/? at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα, and Cox2 particularly in Epm2b ^?/? mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b ^?/? mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD.     

Therapeutic efficacy in BALB/C mice of extract from marine alga <Emphasis Type="Italic">Canistrocarpus cervicornis</Emphasis> (Phaeophyceae) against herpes simplex virus type 1

Studies with diterpenes from marine brown alga Canistrocarpus cervicornis showed the antiviral potential of the products from this alga in controlling the replication of HSV-1 and maintaining low cytotoxicity. Hence, the aim of this work was to evaluate the anti-herpetic efficacy of C. cervicornis extract ointment in BALB/c mice. To test the anti-herpetic efficacy in vivo, four groups of BALB/c mice (n?=?5) were used: 1—untreated, 2—extract ointment (2 % or 0.4 mg cm^?2 dose^?1), 3—Acyclovir cream (5 % or 1.0 mg cm^?2 dose^?1), and 4—ointment base. The right midflank of each mouse was clipped and depilated with a chemical depilatory. After 2 days, the skin area was scratched and inoculated with HSV-1. The ointments and cream were applied three times a day over a 16-day period, beginning 1 h after virus inoculation. The development of skin lesions was continuously monitored and scored. To evaluate the effect of C. cervicornis topical treatment on biochemical parameters and on body weight, two uninfected groups were formed: an untreated group and a group treated with ointment C. cervicornis extract (2 %). The signs of infection appeared from the second day after infection, while on the 10th day of the experiment, the ointment base and untreated groups had significantly more severe lesions than did the groups that were treated with extract (p?<?0.05) or acyclovir (p?<?0,01). The topical application of extract ointment did not change body weight, hepatic, or renal function suggesting that the extract has a low toxicity in this route of administration. These results suggest that the extract may be useful in reducing the severity of HSV-1 cutaneous lesions.     

Polyhydroxyalkanoate Production and Degradation Patterns in <Emphasis Type="Italic">Bacillus</Emphasis> Species

Bacteria under stress conditions of excess of carbon (C) and limitations of nutrients divert its metabolism towards C storage as energy reservoir—polyhydroxyalkanoate (PHA). Different Bacillus species—B. cereus and B. thuringiensis, were monitored to produce PHA from different C sources—glucose, crude glycerol and their combination at 37 °C for period up to 192 h. PHA production and its composition was found to vary with feed and bacterial strains. PHA production on crude glycerol continued to increase up to 120 h, reaching a maximum of 2725 mg/L with an effective yield of 71% of the dry cell mass. Depolymerization of PHA was observe to initiate after 96 h of incubation up to 192 h. PHA degradation products have been envisaged to be applied in medical field: tissue engineering, drug carriers, memory enhancers, antiosteoporosis, biodegradable implants. The PHA production and degradation cycle for 192 h has not been reported previously in literature.     

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> Species

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8?×?10⁶ CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.     

Endocrinopathy in Mice affected with Scrapie

SCRAPIE is a protracted neurologic disease of sheep that is caused by a virus-like agent¹. The prominent neuropathologic lesions of scrapie are hypertrophy and proliferation of astrocytes, vacuolation of grey matter and progressive degeneration of neurones^2,3. The scrapie agent induces a similar disease in the mouse⁴. Characteristic clinical features include a waddling gait, reluctance to move, dullness, loss of condition and stiffness of the tail. Evidence of endocrinopathy was observed in two inbred strains of mice affected with scrapie. I shall describe pathologic changes in the reproductive organs of the Balb/c mouse and change in coat colour of the C57 black mouse.     

The Brain NO Levels and NOS Activities Ascended in the Early and Middle Stages and Descended in the Terminal Stage in Scrapie-Infected Animal Models

The infections of prion agents may cause progressive and fatal neurodegenerative diseases in humans and a serial of animal species. Previous studies have proposed that the levels of nitric oxide (NO) and nitric oxide synthase (NOS) in the brains of some neurodegeneration diseases changed, while S-nitrosylation (SNO) of many brain proteins altered in prion diseases. To elucidate the potential changes of brain NO levels during prion infection, the NO levels and NOS activities in the brain tissues of three scrapie experimental rodents were measured, including scrapie agent 263 K-infected hamsters and 139A- and ME7-infected mice. Both NO levels and NOS activities, including total NOS (TNOS) and inducible NOS (iNOS), were increased at the terminal stages of scrapie-infected animals. Assays of the brain samples collected at different time points during scrapie infection showed that the NO levels and NOS activities started to increase at early stage, reached to the peak in the middle stage, and dropped down at late stage. Western blots for brain iNOS revealed increased firstly and decreased late, especially in the brains of 139A- and ME7-infected mice. In line with those alterations, the levels of the SNO forms of several selected brain proteins such as aquaporin-1 (AQP1), calcium/calmodulin-dependent protein kinase II (CaMKII), neurogranin, and opalin, underwent similar changing trends, while their total protein levels did not change obviously during scrapie infection. Our data here for the first time illustrate the changing profile of brain NO and NOS during prion infection. Time-dependent alterations of brain NO level and the associated protein S-nitrosylation process may contribute greatly to the neuropathological damage in prion diseases.     

Disease phenotype in sheep after infection with cloned murine scrapie strains

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2–4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.     

Prebiotic effects of bovine lactoferrin on specific probiotic bacteria

Bovine lactoferrin (bLf) is a natural iron-binding protein and it has been suggested to be a prebiotic agent, but this finding remains inconclusive. This study explores the prebiotic potential of bLf in 14 probiotics. Initially, bLf (1–32 mg/mL) treatment showed occasional and slight prebiotic activity in several probiotics only during the late experimental period (48, 78 h) at 37 °C. We subsequently supposed that bLf exerts stronger prebiotic effects when probiotic growth has been temperately retarded. Therefore, we incubated the probiotics at different temperatures, namely 37 °C, 28 °C, room temperature (approximately 22–24 °C), and 22 °C, to retard or inhibit their growth. As expected, bLf showed more favorable prebiotic activity in several probiotics when their growth was partially retarded at room temperature. Furthermore, at 22 °C, the growth of Bifidobacterium breve, Lactobacillus coryniformis, L. delbrueckii, L. acidophilus, B. angulatum, B. catenulatum, and L. paraplantarum were completely blocked. Notably, these probiotics started regrowing in the presence of bLf (1–32 mg/mL) in a significant and dose-dependent manner. Accordingly, bLf significantly increased the growth of Pediococcus pentosaceus, L. rhamnosus, and L. paracasei (BCRC 17483; a locally isolated strain) when their growth was retarded by incubation at 22 °C. In conclusion, bLf showed inconsistent prebiotic activity in the 14 probiotics at 37 °C, but revealed strong prebiotic activity in 10 probiotic strains at 22 °C. Therefore, this study enables determining additional roles of Lf in probiotic strains, which can facilitate developing novel combinational approaches by simultaneously using Lf and specific probiotics.     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

The <Emphasis Type="Italic">angora</Emphasis> gene weakens the effect of interaction of the mutant genes <Emphasis Type="Italic">wellhaarig</Emphasis> and <Emphasis Type="Italic">waved alopecia</Emphasis> in mice

The interaction of the mutant genes wellhaarig (we) and waved alopecia (wal) in mice was earlier demonstrated in our laboratory. The we gene significantly accelerates the appearance of alopecia in double we/wewal/wal homozygotes as compared to that in single +/+wal/wal homozygotes. It has been found in this work that the mutant gene angora-Y (Fgf5 ^go-Y ) weakens the effect of interaction of the we and wal genes. The first signs of alopecia appear in mice of the we/wewal/wal genotype at the age of 14 days, in triple Fgf5 ^go-Y /Fgf5 ^go-Y we/wewal/wal homozygotes alopecia is observed seven days later, i. e., in 21-day-old animals. The progression of alopecia in triple homozygotes is expressed to a lesser degree than in double +/+we/wewal/wal homozygotes. A single dose of the Fgf5 ^go-Y gene also decreases the effect of interaction of the we and wal genes, but less than a double dose of this gene. The first signs of alopecia in mice of the +/Fgf5 ^go-Y we/wewal/wal genotype appear only three days later than in double +/+we/wewal/wal homozygotes. The data obtained demonstrate that the Fgf5 ^go-Y gene is a powerful modifier of mutant genes determining the process of alopecia.