Microarray analysis in caudal medulla of cattle orally challenged with bovine spongiform encephalopathy
L.M. Almeida1,2, U. Basu1, J.L. Williams3, S.S. Moore1 and L.L. Guan1
1Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, Canada
2Universidade Estadual de Goiás, Ipameri, GO, Brasil
3Parco Technologico Padano, Polo Universitario, Lodi, Italy
Corresponding author: L.M. Almeida
Genet. Mol. Res. (2011) Ahead of Print
Received August 8, 2011
Accepted September 13, 2011
Published October 25, 2011
Key words: Microarray; Transmissible spongiform encephalopathy; PrionBSE; Orally challenged
Bovine spongiform encephalopathy (BSE) is a fatal disorder in cattle characterized by progressive neurodegeneration of the central nervous system. We investigated the molecular mechanisms involved in neurodegeneration during prion infection through the identification of genes that are differentially expressed (DE) between experimentally infected and non-challenged cattle. Gene expression of caudal medulla from control and orally infected animals was compared by microarray analysis using 24,000 bovine oligonucleotides representing 16,846 different genes to identify DE genes associated with BSE disease. In total, 182 DE genes were identified between normal and BSE-infected tissues (>2.0-fold change, P < 0.01); 81 DE genes had gene ontology functions, which included synapse function, calcium ion regulation, immune and inflammatory response, apoptosis, and cytoskeleton organization; 13 of these genes were found to be involved in 26 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression of five DE genes associated with synapse function (tachykinin, synuclein, neuropeptide Y, cocaine, protein 25 kDa) and three DE genes associated with calcium ion regulation (parvalbumin, visinin-like, and cadherin) was further validated in the medulla tissue of cattle at different infection times (6, 12, 42, and 45 months post-infection) by qRT-PCR. These data will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species.
Bovine Spongiform Encephalopathy (BSE), an infectious neurodegenerative disease in cattle, is one of the fatal transmissible sponge encephalopathies (TSEs) that have been identified in many mammalian species including humans. These diseases are characterized by neurodegeneration and aggregation of aberrantly folded prion protein (Aguzzi et al., 2008). While the physiological changes associated with TSE disease in the brain are well documented, the underlying molecular events involved in neurodegeneration are poorly defined. Some studies have shown that the probability of infection and the outcome of the disease are genetically controlled (Prusiner and Scott, 1997). In sheep, mice and human, a large part of the natural susceptibility to TSE depends on alleles of the PRPN gene (Moreno et al., 2003). However, in cattle the association between Prpn variations and disease incidence is less well defined (Juling et al., 2006), which suggests that genes other than PRPN may be involved in susceptibility of cattle to BSE. Quantitative trait loci (QTL) studies have identified genomic regions associated with disease susceptibility, on Bos tauros chromosomes (BTA) 5, 10, and 20 (Hernandez-Sanches et al., 2002); BTA1, -6, -13, -17, -19 and -X (Zhang et al., 2004); and BTA2, -14, -16, -20, -21 and 28 (Murdoch et al., 2010).
In addition to QTL analysis, other approaches have been used to investigate the molecular mechanisms involved in neurodegeneration associated with prion diseases by identification of genes differentially expressed (DE) between normal and infected tissues These studies have detected differential gene expression by the analysis of cDNA libraries (Diedrich et al., 1991), mRNA differential display (Dandoy-Dron et al., 1998), suppression subtractive hybridization (Kopacek et al., 2000) and more recently using microarrays (Riemer et al., 2004; Greenwood et al., 2005; Sorensen et al., 2008). These studies have revealed multiple genes and signaling pathways that may be involved in TSE pathogenesis.
Recently, we reported 101 DE genes between normal and BSE-infected Peyer’s patch tissues (Khaniya et al., 2009) and 966 DE genes in medulla (Almeida et al., 2011) in cattle orally infected with BSE agent 12 and 45 months post-infection. In the present study, a pooling approach was used in order to analyze a large number of animals and to decrease the individual variation. In total 182 DE genes were identified, from which 45% had previously been detected in a comparison between controls and both 12- and 45-month post-infection animals. Differences in gene expression associated with synapse function and calcium ion regulation were validated by qRT-PCR. Global gene expression analyses through identification of genes DE in response to BSE will help to understand the disease process and may result in the discovery of biomarkers for disease progression, therapeutic targets and elucidate the mechanisms of neuropathology and prion replication.
MATERIAL AND METHODS
BSE challenge of cattle Two hundred steers were selected randomly from farms with no history of BSE. These steers were randomly allocated to 2 groups of about 90 individuals in each group. Both groups were housed at the same location, but separately, in the same barn, and managed under the same conditions. The first group remained as unexposed controls; the second group was exposed orally at approximately 6 months of age in August and September 1998, with 100 grams of BSE brain homogenate with a titre of 103.1 mouse (i.c./i.p.) units LD50/g. Infected and control steers slaughtered at similar time points post-infection (PI) and tissues for transcriptome analysis were snap frozen in liquid nitrogen rapidly following slaughter. RNA was extracted from caudal medulla tissue of animals 12, 18, 40, 45 and 61 months PI in the control group, and in animals at 6, 9, 12, 42 and 45 months PI in the infected group. The challenged animals were tested for signs of disease by immunohistochemistry test (IHC) analysis of obex and medulla tissues at slaughter. Control animals did not display clinical signs of BSE and IHC test of medulla tissues were negative for PrPBSE, while infected animals at 42 and 45 PI showed possible pathological signs of BSE (nervousness, hunched posture, hindlimb paresis) and IHC tests were positive for medulla and obex, confirming the BSE status of these animals.
snip...SEE FULL TEXT ;
Friday, September 23, 2011
Bovine spongiform encephalopathy associated insertion/deletion polymorphisms of the prion protein gene in the four beef cattle breeds from North China
LET'S take a closer look at this new prionpathy or prionopathy, and then let's look at the g-h-BSEalabama mad cow. This new prionopathy in humans? the genetic makeup is IDENTICAL to the g-h-BSEalabama mad cow, the only _documented_ mad cow in the world to date like this, ......wait, it get's better. this new prionpathy is killing young and old humans, with LONG DURATION from onset of symptoms to death, and the symptoms are very similar to nvCJD victims, OH, and the plaques are very similar in some cases too, bbbut, it's not related to the g-h-BSEalabama cow, WAIT NOW, it gets even better, the new human prionpathy that they claim is a genetic TSE, has no relation to any gene mutation in that family. daaa, ya think it could be related to that mad cow with the same genetic make-up ??? there were literally tons and tons of banned mad cow protein in Alabama in commerce, and none of it transmitted to cows, and the cows to humans there from ??? r i g h t $$$
ALABAMA MAD COW g-h-BSEalabama In this study, we identified a novel mutation in the bovine prion protein gene (Prnp), called E211K, of a confirmed BSE positive cow from Alabama, United States of America. This mutation is identical to the E200K pathogenic mutation found in humans with a genetic form of CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. We hypothesize that the bovine Prnp E211K mutation most likely has caused BSE in "the approximately 10-year-old cow" carrying the E221K mutation.
Saturday, August 14, 2010
BSE Case Associated with Prion Protein Gene Mutation (g-h-BSEalabama) and VPSPr PRIONPATHY (see mad cow feed in COMMERCE IN ALABAMA...TSS)
her healthy calf also carried the mutation (J. A. Richt and S. M. Hall PLoS Pathog. 4, e1000156; 2008).
This raises the possibility that the disease could occasionally be genetic in origin. Indeed, the report of the UK BSE Inquiry in 2000 suggested that the UK epidemic had most likely originated from such a mutation and argued against the scrapierelated assumption. Such rare potential pathogenic PRNP mutations could occur in countries at present considered to be free of BSE, such as Australia and New Zealand. So it is important to maintain strict surveillance for BSE in cattle, with rigorous enforcement of the ruminant feed ban (many countries still feed ruminant proteins to pigs). Removal of specified risk material, such as brain and spinal cord, from cattle at slaughter prevents infected material from entering the human food chain. Routine genetic screening of cattle for PRNP mutations, which is now available, could provide additional data on the risk to the public. Because the point mutation identified in the Alabama animals is identical to that responsible for the commonest type of familial (genetic) CJD in humans, it is possible that the resulting infective prion protein might cross the bovine-human species barrier more easily. Patients with vCJD continue to be identified. The fact that this is happening less often should not lead to relaxation of the controls necessary to prevent future outbreaks.
Malcolm A. Ferguson-Smith Cambridge University Department of Veterinary Medicine, Madingley Road, Cambridge CB3 0ES, UK e-mail: email@example.com Jürgen A. Richt College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, Kansas 66506-5601, USA NATURE|Vol 457|26 February 2009
P.9.21 Molecular characterization of BSE in Canada
Jianmin Yang1, Sandor Dudas2, Catherine Graham2, Markus Czub3, Tim McAllister1, Stefanie Czub1 1Agriculture and Agri-Food Canada Research Centre, Canada; 2National and OIE BSE Reference Laboratory, Canada; 3University of Calgary, Canada
Background: Three BSE types (classical and two atypical) have been identified on the basis of molecular characteristics of the misfolded protein associated with the disease. To date, each of these three types have been detected in Canadian cattle.
Objectives: This study was conducted to further characterize the 16 Canadian BSE cases based on the biochemical properties of there associated PrPres.
Methods: Immuno-reactivity, molecular weight, glycoform profiles and relative proteinase K sensitivity of the PrPres from each of the 16 confirmed Canadian BSE cases was determined using modified Western blot analysis.
Results: Fourteen of the 16 Canadian BSE cases were C type, 1 was H type and 1 was L type. The Canadian H and L-type BSE cases exhibited size shifts and changes in glycosylation similar to other atypical BSE cases. PK digestion under mild and stringent conditions revealed a reduced protease resistance of the atypical cases compared to the C-type cases. N terminal- specific antibodies bound to PrPres from H type but not from C or L type. The C-terminal-specific antibodies resulted in a shift in the glycoform profile and detected a fourth band in the Canadian H-type BSE.
Discussion: The C, L and H type BSE cases in Canada exhibit molecular characteristics similar to those described for classical and atypical BSE cases from Europe and Japan. This supports the theory that the importation of BSE contaminated feedstuff is the source of C-type BSE in Canada. *** It also suggests a similar cause or source for atypical BSE in these countries.
STRICTLY PRIVATE AND CONFIDENTIAL 25, AUGUST 1995
To minimise the risk of farmers' claims for compensation from feed compounders.
To minimise the potential damage to compound feed markets through adverse publicity.
To maximise freedom of action for feed compounders, notably by maintaining the availability of meat and bone meal as a raw material in animal feeds, and ensuring time is available to make any changes which may be required.
MAFF remains under pressure in Brussels and is not skilled at handling potentially explosive issues.
5. Tests _may_ show that ruminant feeds have been sold which contain illegal traces of ruminant protein. More likely, a few positive test results will turn up but proof that a particular feed mill knowingly supplied it to a particular farm will be difficult if not impossible.
6. The threat remains real and it will be some years before feed compounders are free of it. The longer we can avoid any direct linkage between feed milling _practices_ and actual BSE cases, the more likely it is that serious damage can be avoided. ...
SEE full text ;
Saturday, November 6, 2010
TAFS1 Position Paper on Position Paper on Relaxation of the Feed Ban in the EU Berne, 2010 TAFS INTERNATIONAL FORUM FOR TRANSMISSIBLE ANIMAL DISEASES AND FOOD SAFETY a non-profit Swiss Foundation
Archive Number 20101206.4364 Published Date 06-DEC-2010
Subject PRO/AH/EDR; Prion disease update 2010 (11)
PRION DISEASE UPDATE 2010 (11)
Wednesday, February 16, 2011
SCRAPIE TRANSMISSION TO CHIMPANZEES
Sunday, April 18, 2010
SCRAPIE AND ATYPICAL SCRAPIE TRANSMISSION STUDIES A REVIEW 2010
Monday, April 25, 2011
Experimental Oral Transmission of Atypical Scrapie to Sheep
Volume 17, Number 5-May 2011
EVIDENCE OF SCRAPIE IN SHEEP AS A RESULT OF FOOD BORNE EXPOSURE
This is provided by the statistically significant increase in the incidence of sheep scrape from 1985, as determined from analyses of the submissions made to VI Centres, and from individual case and flock incident studies. ........
it is clear that the designing scientists must have also shared Mr Bradleyâs surprise at the results because all the dose levels right down to 1 gram triggered infection.
Experimental BSE Infection of Non-human Primates: Efficacy of the Oral Route
Holznagel, E1; Yutzy, B1; Deslys, J-P2; Lasmézas, C2; Pocchiari, M3; Ingrosso, L3; Bierke, P4; Schulz-Schaeffer, W5; Motzkus, D6; Hunsmann, G6; Löwer, J1 1Paul-Ehrlich-Institut, Germany; 2Commissariat à l´Energie Atomique, France; 3Instituto Superiore di Sanità, Italy; 4Swedish Institute for Infectious Disease control, Sweden; 5Georg August University, Germany; 6German Primate Center, Germany
Background: In 2001, a study was initiated in primates to assess the risk for humans to contract BSE through contaminated food. For this purpose, BSE brain was titrated in cynomolgus monkeys.
Aims: The primary objective is the determination of the minimal infectious dose (MID50) for oral exposure to BSE in a simian model, and, by in doing this, to assess the risk for humans. Secondly, we aimed at examining the course of the disease to identify possible biomarkers.
Methods: Groups with six monkeys each were orally dosed with lowering amounts of BSE brain: 16g, 5g, 0.5g, 0.05g, and 0.005g. In a second titration study, animals were intracerebrally (i.c.) dosed (50, 5, 0.5, 0.05, and 0.005 mg).
Results: In an ongoing study, a considerable number of high-dosed macaques already developed simian vCJD upon oral or intracerebral exposure or are at the onset of the clinical phase. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of biomarkers.
Conclusions: Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate. The difference in the incubation period between 5 g oral and 5 mg i.c. is only 1 year (5 years versus 4 years). However, there are rapid progressors among orally dosed monkeys that develop simian vCJD as fast as intracerebrally inoculated animals.
The work referenced was performed in partial fulfilment of the study “BSE in primates“ supported by the EU (QLK1-2002-01096).
Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate.
WE know now, and we knew decades ago, that 5.5 grams of suspect feed in TEXAS was enough to kill 100 cows.
look at the table and you'll see that as little as 1 mg (or 0.001 gm) caused 7% (1 of 14) of the cows to come down with BSE;
Risk of oral infection with bovine spongiform encephalopathy agent in primates
Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.
BSE bovine brain inoculum
100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg
Primate (oral route)* 1/2 (50%)
Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)
RIII mice (ic ip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)
PrPres biochemical detection
The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of bioassays is generally judged to be about plus or minus 1 log. ic ip=intracerebral and intraperitoneal.
Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula
Published online January 27, 2005
Molecular Features of the Protease-resistant Prion Protein (PrPres) in H-type BSE
Biacabe, A-G1; Jacobs, JG2; Gavier-Widén, D3; Vulin, J1; Langeveld, JPM2; Baron, TGM1 1AFSSA, France; 2CIDC-Lelystad, Netherlands; 3SVA, Sweden
Western blot analyses of PrPres accumulating in the brain of BSE-infected cattle have demonstrated 3 different molecular phenotypes regarding to the apparent molecular masses and glycoform ratios of PrPres bands. We initially described isolates (H-type BSE) essentially characterized by higher PrPres molecular mass and decreased levels of the diglycosylated PrPres band, in contrast to the classical type of BSE. This type is also distinct from another BSE phenotype named L-type BSE, or also BASE (for Bovine Amyloid Spongiform Encephalopathy), mainly characterized by a low representation of the diglycosylated PrPres band as well as a lower PrPres molecular mass. Retrospective molecular studies in France of all available BSE cases older than 8 years old and of part of the other cases identified since the beginning of the exhaustive surveillance of the disease in 20001 allowed to identify 7 H-type BSE cases, among 594 BSE cases that could be classified as classical, L- or H-type BSE. By Western blot analysis of H-type PrPres, we described a remarkable specific feature with antibodies raised against the C-terminal region of PrP that demonstrated the existence of a more C-terminal cleaved form of PrPres (named PrPres#2 ), in addition to the usual PrPres form (PrPres #1). In the unglycosylated form, PrPres #2 migrates at about 14 kDa, compared to 20 kDa for PrPres #1. The proportion of the PrPres#2 in cattle seems to by higher compared to the PrPres#1. Furthermore another PK-resistant fragment at about 7 kDa was detected by some more N-terminal antibodies and presumed to be the result of cleavages of both N- and C-terminal parts of PrP. These singular features were maintained after transmission of the disease to C57Bl/6 mice. The identification of these two additional PrPres fragments (PrPres #2 and 7kDa band) reminds features reported respectively in sporadic Creutzfeldt-Jakob disease and in Gerstmann-Sträussler-Scheinker (GSS) syndrome in humans.
Thursday, June 23, 2011
Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits
Thursday, July 21, 2011
A Second Case of Gerstmann-Sträussler-Scheinker Disease Linked to the G131V Mutation in the Prion Protein Gene in a Dutch Patient Journal of Neuropathology & Experimental Neurology:
August 2011 - Volume 70 - Issue 8 - pp 698-702
Saturday, June 25, 2011
Transmissibility of BSE-L and Cattle-Adapted TME Prion Strain to Cynomolgus Macaque
"BSE-L in North America may have existed for decades"
PLEASE NOTE *
Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.
The rancher was a ''dead stock'' feeder using mostly (95 %) downer or dead dairy cattle...
Monday, September 26, 2011
Variably Protease-Sensitive Prionopathy, Prionpathy, Prionopathy, FFI, GSS, gCJD, hvCJD, sCJD, TSE, PRION, update 2011
Wednesday, November 09, 2011
Case report Sporadic fatal insomnia in a young woman: A diagnostic challenge: Case Report TEXAS
HOW TO TURN A POTENTIAL MAD COW VICTIM IN THE USA, INTO A HAPPENSTANCE OF BAD LUCK, A SPONTANEOUS MUTATION FROM NOTHING.
OR WAS IT $$$
This is an interesting editorial about the Mad Cow Disease debacle, and it's ramifications that will continue to play out for decades to come ;
Monday, October 10, 2011
EFSA Journal 2011 The European Response to BSE: A Success Story
EFSA and the European Centre for Disease Prevention and Control (ECDC) recently delivered a scientific opinion on any possible epidemiological or molecular association between TSEs in animals and humans (EFSA Panel on Biological Hazards (BIOHAZ) and ECDC, 2011). This opinion confirmed Classical BSE prions as the only TSE agents demonstrated to be zoonotic so far but the possibility that a small proportion of human cases so far classified as "sporadic" CJD are of zoonotic origin could not be excluded. Moreover, transmission experiments to non-human primates suggest that some TSE agents in addition to Classical BSE prions in cattle (namely L-type Atypical BSE, Classical BSE in sheep, transmissible mink encephalopathy (TME) and chronic wasting disease (CWD) agents) might have zoonotic potential.
see follow-up here about North America BSE Mad Cow TSE prion risk factors, and the ever emerging strains of Transmissible Spongiform Encephalopathy in many species here in the USA, including humans ;
kind regards, terry