11.05.2010: Cryptosporidium, a major cause of diarrhoeal disease
Cryptosporidium and Giardia are the most common cause of protozoal diarrhea worldwide. One to ten Cryptosporidiumm oocysts or Giardia cysts are sufficient to get sick. Cryptosporidium oocysts and Giardia cysts can resist to different physical and chemical treatments. In water, they can persist in different forms a few months. Contamination of foods occur by unhygienic food handling or by the environment. Cryptosporidium oocysts and Giardia cysts can survive to different food process.

Cryptosporidium and Giardia present anthroponotic, zoonotic transmission, waterborne and foodborne transmission. The oocysts and cysts can survive for weeks to months in the environment. Faeces that contain oocysts or cysts may be spread to foods by irrigation or by direct contact. Routine treatments of drinking water eliminate only a fraction of these stages.

The detection by immunofluorescence methods like ELISA or by microscopy method are not
recommended due to genetic diversity, lack of sensitivity and subjectivity of the analyse. Molecular
techniques (real time RT-PCR) are the methods of choice for Cryptosporidium and Giardia detection after DNA extraction and purification from the sample, and special commercial kits were developed.  [18]

Molecular tools improving detection and understanding of Cryptosporidium infections [1]
A review conducted by Xiao 2009 highlights molecular tools detecting and differentiating Cryptosporidium at the species/genotype and subtype levels in humans and animals. These molecular epidemic studies show the importance of Cryptosporidium infections and improves the understanding of their sources. The author stresses the importance of genotyping and subtyping tools to improve the understanding of the epidemiology of cryptosporidiosis.

Understanding of the different metabolic pathways of Plasmodium and Cryptosporidium [2]
According to Mogi and Kita 20101 the Apicomplexans, obligate intracellular parasites, have undergone a reductive evolution like the Plasmodium, which in the blood stages of mammalian hosts, has its mitochondrial enzymes down-regulated and its energy metabolism relies mainly on glycolysis. Mitosomes of Cryptosporidium parvum and Cryptosporidium hominis (in humans) lack mtDNA and other metabolic pathways, which are still present in mitosomes of Cryptosporidium muris (in rodents). The authors write further that Cryptosporidium and Perkinsus use pyruvate-NADP(+) oxidoreductase (PNO), malate-quinone oxidoreductase(MQO), and alternative oxidase (AOX). All apicomplexan parasites and dinoflagellates share MQO. The authors hope that understanding mitochondrial metabolic pathways of Plasmodium, Cryptosporidium and Perkinsus will help the development of new chemotherapeutics

Detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium [3]
Pangansa and colleagues 2010 assessed the effectiveness of a PCR-based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium. This gene displays substantial intraspecific variability in sequence, particularly in a TCA microsatellite region, and is used as a marker in molecular epidemiological studies of Cryptosporidium hominis and Cryptosporidium parvum. REF is useful to detect the nucleotide variability in the gp60 gene within each of the two species. The authors highlight the high-throughput potential and relatively low-cost of REF for genetic analyses of C. hominis and C. parvum, and to identifying sources of infection. REF may also be useful to study other protozoan and metazoan parasites.

Cryptosporidium, the the major cause of dysentery [4]
According to Jex and Gasser 2010 the prevention of cryptosporidiosis in humans should focus on prevention and control strategies and epidemiology using the 60-kDa glycoprotein gene (gp60) as genetic marker for Cryptosporidium infections. The authors provide a global analysis which reveals a low diversity of Cryptosporidium, however, there is a limited knowledge about the genetics of cryptosporidiosis in developing nations in Africa and Asia, and about many animals infection sources. The authors call for comparative genome sequence surveys based on gp60 data to improve intervention strategies against cryptosporidiosis.

Importance of specific methods to detect diarrhoea agents among tourists [5]
Agnamey and colleagues 2010 report diarrhoea among tourists returning from West Africa due to Cryptosporidium hominis and Isospora belli. The authors stress the importance of the detection of coccidiosis in diarrhoeic travellers with the use of specific methods.

New Cryptosporidium parvum subgenotype IIn in Indian children [6]
Analysing diarrhoeal stools from hospitalized Indian children aged <5 years Agnamey and colleagues 2010 found that 2.7% were positive for Cryptosporidium. Microscopy, PCR-RFLP and/or sequencing at the SSU rRNA and Cpgp40/15 loci for species determination and subgenotyping were used. Cryptosporidium hominis was the most frequent genus and subgenotypes Ie, Ia, Ib and Id was spread over all places. The authors report a novel Cryptosporidium parvum subgenotype, IIn. The rate of cryptosporidiosis was reported to increase during hotter and drier weather.

Norway sheep are reservoir of zoonotic Cryptosporidium [7]
Robertson and colleagues 2010 assessed cryptosoridiosis and giardiasis of Norwegian lamb farms using immunofluorescent antibody test detecting Cryptosporidium oocysts and Giardia cysts. Findings of Giardia were 31% and 24% for Cryptosporidium. PCR analysis targeted glutamate dehydrogenase and beta-giardin genes for Giardia, and SSU rRNA and actin genes for Cryptosporidium. Only one isolate of Giardia was Assemblage B (zoonotic), but 35 isolates of Cryptosporidium were cervine genotype (potentially zoonotic). The authors concluded that sheep in Norway are a reservoir of zoonotic Cryptosporidium, but Giardia are of no concern.

Genotypes of Cryptosporidium parvum from calves and sheep in Spain [8]
In Galicia, Spain 49.2% of diarrhoeic calves and 30.7% lambs were tested positive for Cryptosporidium by microscopy and molecular tests. Cryptosporydium parvum from calves belonged to the subtype IiaA15G2R1, and one was of the novel subtype IIaA13G1R1. In sheep the subtipes IiaA16G3R1 and IIaA15G2R1suggest a limited genetic diversity in calves. The authors stress that calves and lambs should be considered as zoonotic reservoirs.

Seasonal variation of the genetic morfology of Cryptosporidium with implication on parasite control in preweaned cattle [9]
Szonyi and colleagues 2010 report that 5% Cryptosporidium parvum-like species and 1% Cryptosporidium andersoni were found using the flotation method. C. parvum-like species was found in 26% of the samples among preweaned calves in summer, compared with 11% in winter, and no oocysts of C. parvum-like in cattle older than 5 months were detected. The 18s rRNA gene revealed that in the summer, 42% of the C. parvum-like oocysts were zoonotic, compared with >74% during the rest of the year. Better understanding of management practices or ecological factors will improve the control of this parasite in preweaned calves say the authors.

Cattle may be a source of Cryptosporidium in Hungary [10]
Plutzer and Karanis 2007 determined the genotype and subtypes of Cryptosporidium from faecal samples from calves with diarrhoea in Hungary Genomic DNA was extracted and nested PCR amplified the partial SSU rRNA and GP60 genes digested by SspI, VspI and MboII restriction enzymes. Cryptosporidium parvum IiaA16G1R1 was found to be the most common subtype and two isolates were found to contain the C. parvum allele IId and a new Cryptosporidium parvum IIa A18G1R1 subgenotype. The authors concluded that cattle can be a source of cryptosporidial infections for humans and animals in Hungary.

Cryptosporidium detection method in water [11]
Nichols, Campbell and Smith 2007 presented a nested PCR-restriction fragment length polymorphism (RFLP) method for the detection Cryptosporidium spp. oocysts in natural mineral waters and drinking waters, detecteding less than 5 oocysts per sample.

Transmission of Cryptosporidium from cattle to man an dairy farms [12]
Khan and colleagues 2010 assessed the importance of animals as source of human Cryptosporidiosis in West Bengal, India. A total of 11.7% of the cattle were found positive for Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae, Cryptosporidium andersoni and Cryptosporidium suis-like genotype. Infection of farm workers comprised Cryptosporidium hominis, C. parvum and a novel C. bovis genotype. The authors highlight the risk of Cryptosporidium transmission from cattle to humans on dairy farms.

Cryptosporidium oocysts in paddock and forage [13]
According to Boyer and Kuczynska 2010 adult beef cattle shed oocysts into the environment. The oocysts of Cryptosporidium oocysts are often found in streams and groundwater in livestock agriculture areas. Mean annual oocyst prevalences on forage were 52.4% in pasture and hay paddocks 40.5%. Wild animals act as vector among paddocks. The authors suggest canopy management, short-cycle rotational grazing, and control of wildlife as strategies to reduce number of Cryptosporidium oocysts in pasture and protect water supplies.

Methods for the diagnosis of Cryptosporidium and Giardia [14]
Cacciò 2004 comments a variety of Polymerase Chain Reaction (PCR) assays have been described for Cryptosporidium and Giardia, and post-PCR analyses.

According to the author PCR does not provide information on the viability and infectivity of these pathogens. To overcome this limitation inclusion/exclusion assays using vital dyes or the Reverse-Transcriptase PCR (RT-PCR) are used. RT-PCR relies on the integrity of the short half-life (seconds) of mRNA to assess the viability of the oocysts. The target is the heat shock protein (hsp) 70 gene. When oocysts are exposed to heat hsps are synthesized providing information about the level of amplification and are an index of viability. Real-time PCR allows the continuous monitoring of amplicon formation throughout the reaction, and quantitative aspect of the infection can be determined.

These methods allow the detection of viability, quantify oocysts/cysts present in a sample, and gives informations about the gene expression during the infection.

Modified method for purifying Cryptosporidium oocysts [15]
Huang and colleagues 2010 describe a modification of the purification method of Cryptosporidium oocysts. They used Sheather's sucrose solution diluted with distilled water as an alternative to PBS. A high viability of the oocysts of more that 96% after 1 hour incubation in 37 degrees C were attained.

Parasitic contamination in wastewater and sludge samples in Tunisia [16]
According to Khouja and colleagues 2010 water scarcity compel to the reuse of wastewater or sludge in Tunisia. Wastewater guidelines specific limits for ova of helminths less than 1 egg/l. Protozoan parasite contamination is not specified. The authors assessed, therefore the Tunisian water treatment and found a high diversity of helminth and protozoa contamination of raw wastewater. Six of eight treated wastewater presented parasite contamination with helminths, Cryptosporidium, Giuardia and Entamoeba. Sludge samples presented parasites, Cryptosporidium and Giardia in high frequence from human and animal origin. The authors stress the importance to monitor wastewater and sludge concerning these pathogens.

Monitoring wastewater plants in China [17]
The removal of Cryptosporidium, Giardia, and microbial indicators, including somatic coliphages and faecal coliforms by treatment plants in Beijing, China presented reduction ratios of 0.12 log for Cryptosporidium and 0.18 log for Giardia by the primary treatment process. The authors note further that oxidation ditch process had higher reduction efficiency to Cryptosporidium and Giardia than anaerobic-anoxic-oxic process and conventional activated sludge process, due to longer retention time and higher concentration.
As tertiary treatment greater reduction of pathogens were attained using membrane ultrafiltration, compared with conventional flocculation sedimentation and sand filtration process, as the tertiary treatment.

[1] Xiao L: Molecular epidemiology of cryptosporidiosis: an update.Exp Parasitol. 2010 Jan;124(1):80-9. Epub 2009 Apr 7.
http://www.ncbi.nlm.nih.gov/pubmed/19358845

[2] Mogi T, Kita K: Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium. Parasitol Int. 2010 Apr 27.
http://www.ncbi.nlm.nih.gov/pubmed/20433942

[3] Pangasa A, Jex AR, Nolan MJ, Campbell BE, Haydon SR, Stevens MA, Gasser RB: Highly sensitive non-isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes infective to humans, and its implications. Electrophoresis. 2010 Apr 23.

http://www3.interscience.wiley.com/journal/123373630/abstract

[4] Jex AR, Gasser RB: Genetic richness and diversity in Cryptosporidium hominis and C. parvum reveals major knowledge gaps and a need for the application of "next generation" technologies-research review. Biotechnol Adv. 2010 Jan-Feb;28(1):17-26.
http://www.ncbi.nlm.nih.gov/pubmed/19699288

[5] Agnamey P, Djeddi D, Oukachbi Z, Totet A, Raccurt CP: Cryptosporidium hominis and Isospora belli diarrhea in travelers returning from West Africa. J Travel Med. 2010 Mar 1;17(2):141-2.
http://www.ncbi.nlm.nih.gov/pubmed/20412184

[6] Ajjampur SS, Liakath FB, Kannan A, Rajendran P, Sarkar R, Moses PD, Simon A, Agarwal I, Mathew A, O'Connor R, Ward H, Kang G: Multi-site Study of Cryptosporidiosis in Indian Children with Diarrhea. J Clin Microbiol. 2010 Apr 14
http://www.ncbi.nlm.nih.gov/pubmed/20392919

[7] Robertson LJ, Gjerde BK, Furuseth Hansen E: The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: A longitudinal investigation of 6 flocks of lambs. Vet Parasitol. 2010 Mar 17.
http://www.ncbi.nlm.nih.gov/pubmed/20381251

[8] Diaz P, Quilez J, Chalmers RM, Panadero R, Lopez C, Sanchez-Acedo C, Morrondo P, Diez-Banos P: Genotype and subtype analysis of Cryptosporidium isolates from calves and lambs in Galicia (NW Spain). Parasitology. 2010 Apr 12:1-7.
http://www.ncbi.nlm.nih.gov/pubmed/20380767

[9] Szonyi B, Bordonaro R, Wade SE, Mohammed HO: Seasonal variation in the prevalence and molecular epidemiology of Cryptosporidium infection in dairy cattle in the New York City Watershed. Parasitol Res. 2010 Apr 16.
http://www.ncbi.nlm.nih.gov/pubmed/20397026

[10] Plutzer J, Karanis P: Genotype and subtype analyses of Cryptosporidium isolates from cattle in Hungary. Vet Parasitol. 2007 May 31;146(3-4):357-62. Epub 2007 Mar 27.
http://www.ncbi.nlm.nih.gov/pubmed/17391853

[11] Nichols RA, Campbell BM, Smith HV: Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay. Appl Environ Microbiol. 2003 Jul;69(7):4183-9.
http://www.ncbi.nlm.nih.gov/pubmed/12839797

[12] Khan SM, Debnath C, Pramanik AK, Xiao L, Nozaki T, Ganguly S: Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India. Vet Parasitol. 2010 Mar 11.
http://www.ncbi.nlm.nih.gov/pubmed/20356678

[13] Boyer DG, Kuczynska E: Prevalence and Concentration of Cryptosporidium Oocysts in Beef Cattle Paddock Soils and Forage. Foodborne Pathog Dis. 2010 Mar 30.
http://www.ncbi.nlm.nih.gov/pubmed/20353289

[14] Cacciò SM: New methods for the diagnosis of Cryptosporidium and Giardia. Parassitologia. 2004 Jun;46(1-2):151-5.
http://www.ncbi.nlm.nih.gov/pubmed/15305706

[15] Huang L, An CX, Zhang SM, Ning CS, Zhang LX: Modified method for purifying Cryptosporidium oocysts. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Feb;28(1):79-80. Chinese.
http://www.ncbi.nlm.nih.gov/pubmed/20411762

[16] Khouja LB, Cama V, Xiao L: Parasitic contamination in wastewater and sludge samples in Tunisia using three different detection techniques. Parasitol Res. 2010 Mar 30.

http://www.ncbi.nlm.nih.gov/pubmed/20352447

[17] Fu CY, Xie X, Huang JJ, Zhang T, Wu QY, Chen JN, Hu HY: Monitoring and evaluation of removal of pathogens at municipal wastewater treatment plants. Water Sci Technol. 2010;61(6):1589-99.
http://www.ncbi.nlm.nih.gov/pubmed/20351439

[18] Ceeram Cryptosporidium Giargia tools. Ceeram publication.
http://www.ceeramtools.com/pdf/ceeram-tools.pdf

                09.05.2010: Brachyspira digestve infections in pigs and birds
Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia are helicoidal Gram negative, mobile and anaerobic bacteria. Brachyspira are responsible for diverse digestive infections in animals and humans, causing economic losses due to the delay of growth of the infected animals.
These bacteria are responsible for the grey diarrhoea syndrome. It is estimated that 70% of the porcine breeding farms are affected by this syndrome.

Detection of Brachyspira [1]
The diagnostic is based on the microorganism isolation by culture followed by its biochemical characterisation, however, the results are not always conclusive. Molecular techniques (real time PCR) are the methods of choice for detecting the presence of the different Brachyspira after extraction and purification of the bacterial DNA.
The molecular detection system Brachyspira Ceeram Tools uses real-time PCR with a detection limit is 5 genome copies. No cross-reaction was observed with other organisms susceptible to infected pigs and in samples of the avian industry.

La, Phillips and Hampson 2003 described a duplex PCR amplifying portions of the Brachyspira hyodysenteriae NADH oxidase gene and the B. pilosicoli 16S rRNA gene for the faecal detection of these spirochaetes. [2]

Comparing ordinary PCR with real time PCR methods to detect Brachyspira [3}
Akase and colleagues 2009 compared ordinary PCR with real time PCR methods to detect Brachyspira infections in swine dysentery cases. The authors found that real time PCR using nox primers was more effective than ordinary PCR and culture.

Genome sequence of Brachyspira hyodysenteriae [4]
Hampson and Ahmed 2009 stressed that the genus Brachyspira contains important gut pathogens of pigs, birds and other animal species, including human beings. The genome sequence of the pathogen Brachyspira hyodysenteriae is characterised by extensive genome adaptation to the environment of the colon. Comparing it with the genome sequences of other Brachyspira may explain their involvement in colitis and diarrhoea.

High prevalence of spirochetosis in cholera patients [5]
Spirochetes from the genus Brachyspira were identified in more than one third of cholera patients in Bangladesh by Nelson and colleagues. The authors recommend that spirochetosis be tracked in cholera outbreaks.

Pathogenicity of Brachyspira pilosicoli [6]
Studies on the physiology of Brachyspira pilosicoli are difficul, because it is an anaerobic spirochete which requires a specialized culture, and its growth is slowly. To study the mechanism of colitis induced by Brachyspira pilosicoli Naresh and colleagues 2009 incubated strains of the bacterium with a human colonic adenocarcinoma cell line (Caco-2 cells).

They found that the effect on the Caco-2 cells was an up-regulation of interleukin-1β (IL-1β) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1β, TNF-α, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. The authors concluded that B. pilosicoli is pathogenic.

Human intestinal spirochetosis [7]
A histological diagnosis of human intestinal spirochetosis were performed inpatients with abdominal pain, bloody stools, diarrhea or bowel symptoms, and in patients which showed only occult fecal blood. PCR analysis found more patients infected with Brachyspira aalborgi than B. pilosicoli. Infestation with B. aalborgi was detected over a 6-year period. Sato and colleaguies 2010 suggest that these spirochetes may be harmless commensals in humans.

Antagonistic effect of gut bacteria on swine enteropathogens [8]
Klose and colleagues 2010 screened beneficial strains from the animal gut to control pathogens related to dysentery in pig, such as Clostridium perfringens type A, various serovars of enterotoxigenic Escherichia coli and Salmonella enterica, as well as Brachyspira pilosicoli.

The authors report that Enterobacteriaceae were effectively inhibited by Lactobacillus salivarius and Lactobacillus reuteri strains. Lactobacillus mucosae, Lactobacillus amylovorus and Bifidobacterium thermophilum, were less effective and their effect was based on the production of organic acid.

The Bacillus subtilis strain was found to have anti-clostridial and anti-Brachyspira pilosicoli effect and homofermentative lactobacilli and Bifidobacterium thermophilum could suppress the growth of Brachyspira pilosicoli. Heterofermentative lactobacilli, such as Lactobacillus reuteri and Lactobacillus mucosae had no effect. Enterococcus faecium and L. amylovorus strain presented antagonism producing lactate and hydrogen peroxide. The authors call for more researches on gut bacteriia as antagonist in pig production.

Probiotic feed additive to inhibit Brachyspira hyodisenteriae which causes dysentery in swine [9]
Close and colleagues isolated strains of Bifidobacterium thermophilum, Enterococcus faecium and Bacillus subtilis which may be used as probiotic feed additives for prevention of swine dysentery. Their characteristics are a well-established identity, antibiotic susceptibility and antagonistic activity against Brachyspira hyodisenteriae.

Probiotic inhibition of Brachyspira hyodisenteriae and Brachyspira pilosicoli [10]
Bernardeau and colleagues 2009 studied the inhibition activity of the probiotic Lactobacillus rhamnosus CNCM-I-3698 and Lactobacillus farciminis CNCM-I-3699 on Brachyspira hyodysenteriae and Brachyspira pilosicoli, agents of Swine Dysentery and Porcine Intestinal Spirochaetosis. The in vitro research demonstrated the trapping of spirochaetal cells in a physical network, and the inhibition of the motility of Brachyspira. The authors call for in vivo studies regarding the use of probiotic lactobacilli as feed addsitive for the prevention of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Human intestinal spirochetosis [11]
According to Tsinganou and Gebbers 2010 Brachyspira aalborgi and Brachyspira pilosicoli predominate in human intestinal spirochetosis. Rates of the disease are low where living standards are high, and common in poor populations. Invasion of spirochetes beyond the surface epithelium may be associated with gastrointestinal symptoms, but is asymptomatic invasion remains at the surface of gut epithelium. Rare cases of spirochetemia and multiple organ failure have been reported in critically ill patients with IS.

Brachyspira murdochii is low pathogenic for pigs [12]
Jensen, Christensen and Boye 2009 describe a Brachyspira murdochii catarrhal colitis in pigs, applying fluorescent in situ hybridization and species-specific oligonucleotide probe targeting 23S rRNA. The bacterium was closely associated with the surface epithelium in diseased pigs. The authors concluded that high numbers of Brachyspira murdochii is low pathogenic for pigs.

Pathogenicity of Brachyspira among poultry [13]
Brachyspira intermedia and Brachyspira pilosicoli were found to be affect layer and breeder flocks in Europe and Australia. Myers and colleagues 2009 tested flocks of Pennsylvania for Brachyspira intermedia and Brachyspira pilosicoli using a duplex PCR and Brachyspira genus-specific PCR. The authors found colonization of layer flocks, older than 40 weeks of age, with pathogenic and call for further investigation.

Brachyspira intermedia and other indole-positive Brachyspira species [14]
Phillips and colleagues 2009 using the multilocus sequence typing assessed the population structure of Brachyspira intermedia isolates from pigs and chicken, and the relationship of the species to the other two indole-positive but strongly haemolytic Brachyspira species-B. hyodysenteriae and "B. suanatina".

The results of sequence types, amino acid types and clonal complexes indicate that cross-species may occur. Some isolates were found to be separated from others by large genetic distances and ongoing minor genetic change amongst isolates at the farm level are taking place. The authors conclude that their findings concerning isolates of B. hyodysenteriae, and "B. suanatina", suggest that it will not be possible to classify all weakly haemolytic indole-positive Brachyspira isolates as Brachyspira intermedia.

Relations between strains of Spanish Brachyspira spp and isolates from Germany and Belgium [15]
Hidalgo and colleagues 2010 described the genetic and phenotypic diversity of Spanish Brachyspira hyodysenteriae, and confirmed the presence of tiamulin-resistant isolates in Spain.

The PCR analysis was used for the identification of Brachyspira spp. and for the detection of the smpA/smpB gene. The combination of Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) protocol was used to determine the epidemiological relationships.

The authors stress that indole-negative and tiamulin-resistant isolates of B. hyodysenteriae were found, and the genetic findings indicated a relationship between Spanish isolates and strains of Germany and Belgium.

[1] Fourrier A, Loisy-Hamon F, Guillaume J-M, Lebeau, B: CeeramTools molecular detection system for the detection of pathogens Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in pigs. Ceeram publication.
http://www.ceeramtools.com/pdf/ceeram-tools.pdf

[2] La T, Phillips ND, Hampson DJ: Development of a duplex PCR assay for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig feces. J Clin Microbiol. 2003 Jul;41(7):3372-5.
http://jcm.asm.org/cgi/content/full/41/7/3372

[3] Akase S, Uchitani Y, Sohmura Y, Tatsuta K, Sadamasu K, Adachi Y: Application of real time PCR for diagnosis of Swine Dysentery. J Vet Med Sci. 2009 Mar;71(3):359-62
http://www.ncbi.nlm.nih.gov/pubmed/19346708

[4] Hampson DJ, Ahmed N: Spirochaetes as intestinal pathogens: Lessons from a Brachyspira genome. Gut Pathog. 2009 May 1;1(1):10.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680911

[5] Nelson EJ, Tanudra A, Chowdhury A, Kane AV, Qadri F, Calderwood SB, Coburn J, Camilli A: High prevalence of spirochetosis in cholera patients, Bangladesh. Emerg Infect Dis. 2009 Apr;15(4):571-3.
http://www.ncbi.nlm.nih.gov/pubmed/19331734

[6] Naresh R, Song Y, Hampson DJ: The intestinal spirochete Brachyspira pilosicoli attaches to cultured Caco-2 cells and induces pathological changes. PLoS One. 2009 Dec 17;4(12):e8352.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791440/

[7] Sato H, Nakamura SI, Habano W, Wakabayashi G, Adachi Y: Human intestinal spirochetosis in northern Japan. J Med Microbiol. 2010 Apr 8.
http://www.ncbi.nlm.nih.gov/pubmed/20378723

[8] Klose V, Bayer K, Bruckbeck R, Schatzmayr G, Loibner AP: In vitro antagonistic activities of animal intestinal strains against swine-associated pathogens. Vet Microbiol. 2010 Feb 20.
http://www.ncbi.nlm.nih.gov/pubmed/20226602

[9] Klose V, Bruckbeck R, Henikl S, Schatzmayr G, Loibner AP: Identification and antimicrobial susceptibility of porcine bacteria that inhibit the growth of Brachyspira hyodysenteriae in vitro. J Appl Microbiol. 2010 Apr;108(4):1271-80.
http://www.ncbi.nlm.nih.gov/pubmed/19778354

[10] Bernardeau M, Gueguen M, Smith DG, Corona-Barrera E, Vernoux JP: In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli. Vet Microbiol. 2009 Jul 2;138(1-2):184-90.
http://www.ncbi.nlm.nih.gov/pubmed/19356863

[11] Tsinganou E, Gebbers JO: Human intestinal spirochetosis-a review. Ger Med Sci. 2010 Jan 7;8:Doc01.
http://www.ncbi.nlm.nih.gov/pubmed/20200654

[12] Jensen TK, Christensen AS, Boye M: Brachyspira murdochii colitis in pigs.Vet Pathol. 2010 Mar;47(2):334-8. Epub 2009 Dec 31.
http://www.ncbi.nlm.nih.gov/pubmed/20173181

[13] Myers SE, Dunn PA, Phillips ND, La T, Hampson DJ: Brachyspira intermedia and Brachyspira pilosicoli are commonly found in older laying flocks in Pennsylvania. Avian Dis. 2009 Dec;53(4):533-7.
http://www.ncbi.nlm.nih.gov/pubmed/20095153

[14] Phillips ND, La T, Amin MM, Hampson DJ: Brachyspira intermedia strain diversity and relationships to the other indole-positive Brachyspira species. Vet Microbiol. 2009 Oct 29.
http://www.ncbi.nlm.nih.gov/pubmed/19944544

[15] Hidalgo A, Carvajal A, Pringle M, Rubio P, Fellström C: Characterization and epidemiological relationships of Spanish Brachyspira hyodysenteriae field isolates. Epidemiol Infect. 2010 Jan;138(1):76-85.
http://www.ncbi.nlm.nih.gov/pubmed/19480724

09.05.2010: Impact of Crude Oil on Seafood, the Horizon oil spill [1]
According to the National Oceanic and Atmospheric Administration (NOAA) crude oil has the potential to taint seafood with flavours and odours imparted by exposure to hydrocarbon chemicals. The U.S. Food and Drug monitors the oil contamination of seafood. The agency says the public should not be concerned about the safety of seafood they are buying at this time.

NOAA conducts a combination of both sensory analysis (of tissue) and chemical analysis (of water, sediment, and tissue) to determine if seafood is safe following an oil spill. The results will be made pubic as soon as possible. This inspection program is based on lessons learned from major oil spills such as the 1989 Exxon Valdez spill in Alaska; 1996 spill in Rhode Island; 1999 spill in Coos Bay, Oregon; and, several spills in San Francisco including the most recent in 2007.

Meanwhile all commercial fishing is prohibited in waters in front of Lousiana. Oil has reached the islands in front of the mainland. The marshland of the Mississippi delta is endangered. [2]

Ocean Studies Board (OSB) rises doubts on the use of dispersants to fight the Horizon oil spill [3]
The Ocean Studies Board (OSB) in 2005 cited possible harms to the environment when oil dispersants are used. Fishes corals, shrimps, oysters and other marine life may be poisoned by the chemicals and the toxic components of the dispersed oil. The use of dispersants reduces the threat posed to marine mammals and birds that frequent the air-water interface where oil slicks form. The dispersed oil plume affects other portions of the ecosystem, such as the fish and the fauna in water column and on the seafloor. Dispersants may also affect the water-repellent layer of the feathers of seabirds.

Dispersants are chemical agents such as surfactants, solvents, and other compounds that reduce interfacial tension between oil and water in order to enhance the natural process of dispersion by generating larger numbers of small droplets of oil which spread in all directions. Oil spill dispersants do not reduce the total amount of oil entering the environment.

The oil spill dispersant Corexit 9500 used at the Horizon spill [4]
The Corexit oil spill dispersant is biodegradable, has low toxicity and enhanced penetration of the
surfactants and helps to counter the “mousse” forming tendencies of the spilled. Its ingredients are distillates, petroleum, hydrotreated light 30%, Propylene Glycol 5% Organic sulfonic acid salt 30%. Sulfonic acids and their salts (sulfonates) are used extensively in such diverse products like detergents. [5]

Hydrogen economy to phase out carbon fuel [6]
A strategy to decarbonise the fuel economy is most important for a sustainable future. Solar energy and wind turbines, together with hydrogen technology is the solution for the environmental harm which is done by exploiting oil, gas, coal and uranium mines.

[1] NOAA Fishery Service: Impact of Crude Oil on Seafood.
http://www.deepwaterhorizonresponse.com/posted/2931/Seafood_safety_FACT_SHEET.542647.pdf

[2] Kampf gegen Oelpest: Umstrittene Chemikalien. Spiegel Online 08.05.2010
http://www.spiegel.de/fotostrecke/fotostrecke-54635-2.html

[3] Oil Spill Dispersants, Efficacy and Effects: Committee on Understanding Oil Spill Dispersants: Efficacy and Effects. Ocean Studies Board. Division on Earth and Life Studies. National Research Council of the National Academies.
http://www.nap.edu/openbook.php?record_id=11283&page=1

[4] Corexit 9500(EC9500A) Oil Spill Dispersant. Nalco Energy Services. Product Bulletin PB–EC9500A
http://www.cleancaribbean.org/docs/9500A%20Product%20Sheet_01_06.pdf

[5] Corexit 9500, NALCO Material Data Sheet
http://www.deepwaterhorizonresponse.com/posted/2931/COREXIT_9500_UsCuEg.539287.pdf

[6] Desert Energy Project.net The Global Hydrogen Initiative.
http://www.desertenergyproject.net/

08.05.2010: High dioxin levels in German organic eggs, failure of incoming controls? [1]
Dioxins increase the risk of cancer. Levels above the EU standard of 3 pg TEQ were found in German organic eggs by the Association for controlled alternative husbandary, which maintains the label: "Controlled by KAT". The whole production chain did not comply with the HACCP and ISO 9000 principles, questioning organic eggs and their seals of approval.

The grocery store chain LIDL removed all dioxin contaminated organic eggs from its shelves.

Investigations found that the organic feed from the Dutch Harreveld facility of ForFarmers were the source of the dioxin. Organic corn for the production of the feed imported from the Ukraine could be traced as the primary source of the contamination. Incoming controls have failed. It is a sign of failure of the quality system when toxic food is found at the end of the chain and hazzards are not identified with incoming controls of the Ukrainian corn. Organic eggs are continuously on the headlines of food scandals. [2]

Belgian dioxin crisis related to chicken feed in 1999 [3]
In 1999, Belgium had a dioxin crisis caused by dioxin-contaminated feed being fed to livestock. The source of the contamination was a Belgian at-rendering company, where transformer oil with high levels of polychlorinated biphenyls (PCBs) and dioxins was used to manufacture animal foods sale of Belgian poultry and eggs and all food items containing >2% egg product

Dioxin levels are higher in eggs of free-ranging chickens than chickens kept inside [4]
De Vries and colleagues 2006 found that dioxin levels are higher in eggs from free-ranging chickens than in eggs from chickens kept inside. Free-ranging chickens ingest soil and eat insects and worms, all of which contain environmental dioxins. Flock size influences the behaviour of the animals. Small flocks are outside most of the time whereas large flocks tend to remain inside. The uptake of dioxin-contaminated soil or insects taken up varies accordingly. The authors say that large Dutch farms with more than 1500 laying hen have egg dioxin levels below the EU standard of 3 pg TEQ, while organic farms with small flocks present unacceptably high egg dioxin levels because the animals spend most of the time outside.

In Lower Saxony state, 28% of all free-range eggs produced in the last two years were above European Union limits for dioxin levels, because hens were allowed to roam on land contaminated with the chemicals. [5]

[1] Rhein-Wied-News: Lidl nimmt dioxinverseuchte Bio-Eier aus dem Handel - auch ein Legehof in RLP geschlossen! Pressemitteilung WS 07.05.2010
http://pressemitteilung.ws/node/208328

[2] Sofort umfangreiche Untersuchungen eingeleitet. Keine Bio-Eier mit erhöhtem Dioxingehalt im Verkauf. Na-Presseportal 05.05.2010.
http://www.presseportal.de/pm/73754/1608864/verein_fuer_kontrollierte_alternative_tierhaltungsformen_e_v_kat

[3] Vellinga A, Van Loock F: The Dioxin Crisis as Experiment To Determine Poultry-Related Campylobacter Enteritis.
http://www.cdc.gov/ncidod/eid/vol8no1/01-0129.htm

[4] De Vries1M, Kwakkel R P, Kijlstra A: Dioxins in organic eggs: a review. NJAS 54-2, 2006
http://library.wur.nl/ojs/index.php/njas/article/viewFile/1161/739

[5] Dioxin found in German eggs. Germany has called for higher environmental standards on farms after free-range eggs were found to contain the cancer causing chemical dioxin. BBC News 17 January 2005.
http://news.bbc.co.uk/2/hi/science/nature/4182029.stm 

                                        06.05.2010: Norovirus
Noroviruses are the most common viral agents of acute gastroenteritis in humans. Noroviruses can genetically be classified into 5 different genogroups (GI, GII, GIII, GIV, and GV), which can be further divided into different genetic clusters or genotypes. For example genogroup II, the most prevalent human genogroup, presently contains 19 genotypes. Genogroups I, II and IV infect humans, whereas genogroup III infects bovine species and genogroup V has recently been isolated in mice.

Many studies on the epidemiological and genetic characteristics of Noroviruses outbreaks point to their importance in food safety. Rapid analytical methods were developed to identify the source of epidemics.

Norovirus GII.4 leading global cause of viral gastroenteris [1]
According to Said, Perl and Sears 2008 the Noroviruses are a leading global cause of viral gastroenteritis and a major contributor to food-borne illness. According to the authors the GII.4 strain of the virus dominates in epidemics by antigenic drift evading thus the immune system.

Norovirus NoV frequent agent of gastroenteritis in an older population [2]
Rosenthal and colleagues 2010 assessing outbreaks infections in an older population found that 70% were caused by Noroviruses, with a case-fatality rate of 0.5%. GII.4 strains accounted for 84% of Noroviruses outbreaks and had a mean duration of 33 hours versus 24 hours of non-GII.4 infections.

GAP, GMP and HACCP improves microbiological safety of ready to eat vegetables [3]
De Giusti and colleagues 2010 report that GAP, GMP and HACCP improves the microbiological safety of ready to eat vegetables compared with different preventive strategies, such as use of chlorine disinfection at a second washing step, or using a physical microbial stabilization. The aerobic mesophilic count and Escherichia coli were quantified, and the presence of Salmonella spp, Listeria monocytogenes, E. coli O157:H7, hepatitis A virus and Norovirus were determined. The authors found that GAP, GMP and HACCP presented better microbiological quality than those processed with chemical or physical stabilization.

Water purification systems are unable to remove Norovirus from bivalve moluscs [4]
The efficacy of the water depuration systems in the presence of Norovirus contamination of bivalve moluscs. Mussels, Manila clam and Pacific oyster were examined by reverse transcriptase-polymerase chain reaction before and after depuration. Viral RNA was detected non-depurated samples as well in depurated samples indicating that the purifying systems in place were not able to remove Norovirus contamination from the live bivalve molluscs, write Savini and colleagues 2009.

Removal of Norovirus during a coagulation-ceramic microfiltration of drinking water [5]
Shirasaki and colleagues 2010 assessed the removal performance as particles during a coagulation-ceramic microfiltration process using recombinant NV virus-like particles (rNV-VLPs), and the bacteriophages Qbeta and MS2, similar to Noroviruses. More than 4-log removal of rNV-VLPs with a 1.08 mg-Al/L dose of polyaluminium chloride in the coagulation-ceramic MF process was found . The removal ratios of Qbeta and MS2 were smaller than the ratio of rNV-VLPs. The authors concluded that both bacteriophages are appropriate surrogates for native Noroviruses in the coagulation-ceramic MF process. They suggest the use of Qbeta as surrogate.

Review of Calicivirus studies [6]
Catpally and colleagues reviewed the current studies of Noroviruses which had been hampered by the lack of animal model and tissue culture system. The authors report that recent advances in protein expression systems and the development of a mouse norovirus animal model have brought a rapid growing knowledge about these viruses.

Wastewater and crop contamination by Norovirus and Ascaris lumbricoides [7]
Mara and Sleigh 2010 estimate norovirus and Ascaris infection risks to urban farmers in developing countries using wastwater for crop irrigation. To achieve a tolerable disease burden of 1 percent of the diarrhoeal disease a norovirus reduction of 1-2 log units and an Ascaris egg reduction to 10-100 eggs per litre are required. The authors stress that such reductions are easily achieved by minimal wastewater. A sequential batch-fed three tank/pond system are being suggested, in addition to education and regular deworming in farming regions.

The authors reminds that reductions of 4-6 log units can be achieved using settling basins (1-log unit), pathogen die-off (1-2 log units), produce washing in cold water (1 log unit) and produce disinfection (3 log units).[8]

[1] Said MA, Perl TM, Sears CL: Healthcare epidemiology: gastrointestinal flu: norovirus in health care and long-term care facilities.Clin Infect Dis. 2008 Nov 1;47(9):1202-8.
http://www.ncbi.nlm.nih.gov/pubmed/18808354

[2] Rosenthal NA, Lee LE, Vermeulen BA, Hedberg K, Keene WE, Widdowson MA, Cieslak PR, Vinjé J: Epidemiological and genetic characteristics of norovirus outbreaks in long-term care facilities, 2003-2006. Epidemiol Infect. 2010 Apr 23:1-9.
http://www.ncbi.nlm.nih.gov/pubmed/20412611

[3] De Giusti M, Aurigemma C, Marinelli L, Tufi D, De Medici D, Di Pasquale S, De Vito C, Boccia A: The evaluation of the microbial safety of fresh ready-to-eat vegetables produced by different technologies in Italy.J Appl Microbiol. 2010 Mar 22
http://www.ncbi.nlm.nih.gov/pubmed/20408920

[4] Savini G, Casaccia C, Barile NB, Paoletti M, Pinoni C: Norovirus in bivalve molluscs: a study of the efficacy of the depuration system. Vet Ital. 2009 Oct-Dec;45(4):535-9.
http://www.ncbi.nlm.nih.gov/pubmed/20391417

[5] Shirasaki N, Matsushita T, Matsui Y, Urasaki T, Oshiba A, Ohno K: Evaluation of norovirus removal performance in a coagulation-ceramic microfiltration process by using recombinant norovirus virus-like particles. Water Sci Technol. 2010;61(8):2027-34.
http://www.ncbi.nlm.nih.gov/pubmed/20389000

[6] Katpally U, Smith TJ: The Caliciviruses. Curr Top Microbiol Immunol. 2010 Apr 1.
http://www.ncbi.nlm.nih.gov/pubmed/20376611

[7] Mara D, Sleigh A: Estimation of norovirus and Ascaris infection risks to urban farmers in developing countries using wastewater for crop irrigation. J Water Health. 2010 Sep;8(3):572-6. Epub 2010 Mar 9.
http://www.ncbi.nlm.nih.gov/pubmed/20375486

[8] Mara D, Sleigh A: Estimation of norovirus infection risks to consumers of wastewater-irrigated food crops eaten raw. J Water Health. 2010 Mar;8(1):39-43.
http://www.ncbi.nlm.nih.gov/pubmed/20009246

27.04.2010: Nicotine dependence
Recent articles report that genetic mutations on chromosomes 8, 15 and 19 make addiction to smoke more critical, and they are more prone to lung cancer compared to persons without this mutation. Also chromosome 11 is known to intensify the addiction, but chromosome 9 contains a gene which make it less difficult to stop smoking.
The researchers say that some genes are responsible for the synthesis of enzymes which may be active in metabolising nicotine. Nicotine activates the nicotine receptors in the brain which releases the "feel good" hormones dopamine and serotonine.

Genes associated with smoking behaviour [1]
The Tobacco and Genetics Consortium together with the European Network of Genetic and Genomic Epidemiology and the Oxford-GlaxoSmithKline identified three loci associated with number of cigarettes smoked per day. These loci were the 15q25 single-nucleotide polymorphism (SNP) in the nicotinic receptor gene CHRNA3, two 10q25 SNPs, and one 9q13 SNP in EGLN2. Other genes were found which facilitate smoking initiation, and other genes which are. The good news are that one loci located on chromosome 9 was significantly associated with smoking cessation.

Smoking behaviour and genetics [2]
Thorgeirsson and colleagues report that variants in the genomic regions at 15q25, 19q13, 8p11, and rs6474412-T are related to number of cigarettes smoked per day. Two loci are genes encoding nicotine-metabolizing enzymes (CYP2A6 and CYP2B6) and nicotinic acetylcholine receptor subunits (CHRNB3 and CHRNA6), already known to be related to smoking and nicotine dependence. The genes at 8p11 and 19q13 are linked with lung cancer

Gene locus on 15q25 associated with smoking quantity [3]
Liu and colleagues 2010 confirmed an effect on smoking quantity at a locus on 15q25 that includes CHRNA5, CHRNA3 and CHRNB4, three genes encoding neuronal nicotinic acetylcholine receptor subunits.

Genetics as culprit to failure to quit smoking? [4]
Chapman and MacKenzie 2010 argue that the volume of research and effort devoted to professionally and pharmacologically mediated cessation is in inverse proportion to that examining how most ex-smokers actually quit.
The authors say that final results of nicotine replacement therapy or other drugs is dramatically overestimated. Many of these studies are funded by pharmaceutical companies.

Up to three-quarters of ex-smokers have quit without assistance, and unaided cessation is by far the most common method used by most successful ex-smokers. Recidivism is normal if efforts are not serious attempts. The increasing medicalisation of smoking cessation implies professionally mediation. The author call on health authorities emphasise the positive message that the most successful method used by most ex-smokers is unassisted cessation. Nicotine replacement therapy may help, but is not used in most cases of successful cessations.

Small hint to quit smoking: The most stringent factor needed to stop smoking is a strong will to do it. The first 4 days of cessation are difficult. You have to avoid any trouble. Take a vacation or some days off for the start. You will feel sick and nervous. Remember that after this period you will feel better each day. You will be proud of your mental strength and you will be able to handle other weaknesses much easier. Do not blame genetics. Pharmaceutical companies, like Oxford-GlaxoSmithKline, pose to much emphasis on studies related to nicotine replacement and genetics. It is your strength which you have to rely on.

[1] The Tobacco and Genetics Consortium: Genome-wide meta-analyses identify multiple loci associated with smoking behavior. Nature Genetics Published online: 25 April 2010. Doi:10.1038/ng.571

http://www.nature.com/ng/journal/vaop/ncurrent/abs/ng.571.html#/

[2] Thorgeirsson et al.: Sequence variants at CHRNB3–CHRNA6 and CYP2A6 affect smoking behavior. Nature Genetics. 25 April 2ß1ß. Doi:10.1038/ng.573
http://www.nature.com/ng/journal/vaop/ncurrent/abs/ng.573.html#/

[3] Liu et al: Meta-analysis and imputation refines the association of 15q25 with smoking quantity. Nature Genetics. 25 April 2010. Doi:10.1038/ng.572
http://www.nature.com/ng/journal/vaop/ncurrent/abs/ng.572.html#/ar

[4] Chapman, Simon; MacKenzie, Ross: The Global Research Neglect of Unassisted Smoking Cessation: Causes and Consequences. PLoS Med 7(2), February 9, 2010: e1000216. doi:10.1371/journal.pmed.1000216
http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1000216