CEEX-AGRAL 74

 Denumirea proiectului: Studii avansate privind posibilitatile de prevenire a imbolnavirilor produse de unele specii toxigene de Fusarium sp. in lantul trofic plante furajere-animal-om

(english version)

Sursa de finantare: Bugetul de Stat – Ministerul Educatiei si Cercetarii, Programul ”Cercetare de excelenta”

Autoritatea contractanta:
Universitatea de Stiinte Agronomice si Medicina Veterinara Bucuresti

Contractor:
Institutul National de Cercetare –Dezvoltare Agricola Fundulea

Categoria de proiect: Modulul 1- Proiecte de Cercetare – Dezvoltare Complexe, tipul de proiect P–CD

Valoarea contractului: 1.500.000 lei - din care:

 

Parteneri:                  

Coordonator proiect: Institutul National de Cercetare – Dezvoltare Agricola – Fundulea, str. N.Titulescu. nr. 1, 915200, Fundulea , tel. +40213154040, fax. +40242642044, E-mail: tina@ricic.ro, web site: www.incda-fundulea.ro
Director proiect: Dr. Ing. Florentina Raducanu

Partener 1: Institutul de Cercetare Dezvoltare pentru Cresterea Ovinelor si Caprinelor Palas Constanta, Soseaua I.C.Bratianu, nr. 248, 060042, Constanta, Tel/fax:0241548186/0241639506, e-mail: stela@canals.ro, www.icdoc.ro Responsabil stiintific: Dr. Dorina Nadolu

Partener 2: Universitatea Ovidius Constanta,str. B-dul Mamaia,nr.124, tel/fax;024169040/02416639506; e-mail:rectorat@univ-ovidius.ro ; www.univ-ovidius.ro
Responsabil stiintific: Prof.univ.dr. Stela Zamfirescu

 

Aria tematică : 2.    Alimentatie, agricultura si biotehnologii: 2.2 Alimentatie, sanatate si buna stare; Aspecte ale hranei si alimentatiei din punctul de vedere al consumatorului, al societatii si al protejarii sanatatii, inclusiv legate de stiintele comportamentale si cognitive; tehnologii inovative privind hrana si prelucrarea alimentelor (inclusiv hrana functionala), imbunatatirea calitatii si sigurantei chimice si microbiologice a alimentelor si bauturilor, integritatea lantului alimentar, lantul alimentar total, inclusiv transabilitatea acestuia.

Rezumatul propunerii: Cercetarile care vor fi efectuate in cadrul acestui proiect, urmaresc  in primul rand obtinerea unor mutante rezistente de Triticale (Triticosecale Wittmack) si  lucerna (Medicago sativa L.) fata de patogenii toxigeni Fusarium graminearum si Fusarium oxysporum, prin utilizarea unor metode si tehnici neconventionale. In acest fel, se are in vedere imbunatatirea intr-un timp relativ scurt, a calitatii si sigurantei chimice si microbiologice a furajelor cu implicatii asupra intregului lant alimentar si asupra trasabilitatii acestuia. In paralel, vor fi efectuate studii aprofundate in vederea evidentierii modificarilor produse de toxina patogenului la nivelul profilelor: biochimic, hematologic si ultrastructural la sobolani alimentati cu lapte si carne, alimente provenite de la oi si capre hranite in exclusivitate cu furaje infestate (triticale si lucerna), in vederea producerii de anticorpi monoclonali pentru detectarea infectiilor cu Fusarium sp., la om. Astfel, intr-o prima etapa, vor fi efectuate studii privind unele componenete ale mecanismelor  defensive la genotipuri de triticale si lucerna (prezenta chitinazei in radacinile si tulpinile plantelor infectate in vivo, cu filtrat de F. oxysporum in conditii  controlate de mediu); in scopul determinarii gradului real de rezistenta al genotipurilor  comparativ cu reactia lor fenotipica. Concomitent, se vor efectua lucrari de testare in vederea selectiei genotipurilor in ceea ce priveste pretabilitatea la cultura in vitro. Genotipurile cu raspuns foarte bun (capacitate de calusare si regenerare) caracterizate si prin insusiri agronomice superioare vor fi utilizate atat pentru studiile efectuate in vitro, cat si pentru infiintarea bazei furajere, care sa asigure hranirea  unui esantion de oi si capre, pe tot parcursul experimentului. Genotipurile selectionate vor fi introduse intr-un ciclu riguros de testare si selectie in vitro, in prezenta micotoxinelor deoxynivalenol DON si  acid fusaric (toxinele predominante ale speciilor de Fusarium luate in studiu) in mediile de cultura. Pe parcursul experimentului vor fi efectuate studii aprofundate cu privire la: i) influenta micotoxinei asupra celulelor vegetale testate in vitro in prezenta acidului fusaric (determinari ale continutului in clorofila, a suprafetei foliare, continutul in prolina, studiul izoenzimelor specifice genotipurilor de la care   s-a pornit experimentul si a celor obtinute in vitro sub influenta stresului indus de prezenta in mediile de cultura a micotoxinelor si al filtratelor de cultura). ii) Vor fi elaborate metode paraclinice de stabilire a nivelului toxinelor in ser, determinari ale compozitiei chimice a laptelui si carnii de ovine si caprine hranite cu furaje infestate. Tinand cont de toxicitatea micotoxinelor prezente in filtratele de cultura, aplicarea tratamentelor se va efectua la nivel de ratie zilnica /cap animal furajat, in conditii controlate, in spatii inchise, luindu-se toate masurile de protectie a muncii pentru personalul care va efectua tratamentele. iii) De asemenea, se vor efectua teste de laborator asupra alimentelor, la inceputul, pe parcursul si la finalul cercetarii in vederea inregistrarii cu exactitate a modificarilor biochimice, atat a produselor lactate, la nivelul organismului animalelor furajate, cat si la nivelul animalelor de laborator  hranite cu lapte si derivatele lui si vor fi efectuate studii electronomicroscopice la nivelul unor parametri metabolici implicati in intoxicarea cu micotoxinele studiate. Rezultatele estimate in urma proiectului sunt pe de o parte, obtinerea unor genotipuri de triticale si lucerna intr-un timp relativ scurt cu rezistenta sporita la atacul de Fusarium graminearum si Fusarium oxysporum, care vor asigura obtinerea de furaj de calitate, liber de toxine (plante pentru viitor)  iar pe de alta parte, rezultate stiintifice bine fundamentate privind posibilitatile de izolare a micotoxinelor si de limitare a influentei acestora asupra intregului lant trofic: planta infectata-animal furajat cu furajul infectat-animal hranit (sobolani) cu laptele si derivatele sale provenit de la animalele furajate exclusiv cu furaj infestat. Vor fi elucidate aspecte legate de timpul de metabolizare a acidului fusaric la nivel de planta, produs animalier si organismul animalului de laborator in vederea producerii de anticorpi monoclonali pentru detectarea infectiilor cu Fusarium sp. la om.

OBIECTIVE GENERALE

  1. Inducerea variabilitatii somaclonale prin embriogeneza somatica indirecta la genotipuri de triticale si lucerna  cu insusiri agronomice superioare.
  2. Elaborarea unor metodologii de testare si selectie, cu randament ridicat al capacitatii de regenerare (parametru deficitar la culturile in vitro).
  3. Identificarea unei metode eficiente de obtinere a izolatelor toxigene si a filtratelor de Fusarium sp.
  4. Identificarea modificarilor survenite la plante regenerate pe medii suplimentate cu filtrate de Fusarium sp. si /sau micotoxinele DON si acid fusaric.
  5. Obtinerea de somaclone tolerante/rezistente la Fusarium graminearum si Fusarium oxysporum.
  6. Identificarea modificarilor biochimice, hematologice si ultrastructurale la animalele hranite cu furaje infestate cu Fusarium sp.
  7. Elaborarea unei metode  paraclinice pentru stabilirea nivelului de toxine din ser si lapte.
  8. Identificarea modificarilor produse de micotoxinele patogenului Fusarium sp.asupra compozitiei chimice a produselor animaliere.
  9. Identificarea efectelor micotoxinelor patogenului Fusarium sp. asupra reproductiei.
  10. Elaborarea unui test imunologic pentru diagnosticarea la om a intoxicatiilor produse de micotoxine.

PROBLEME PROPUSE SPRE REZOLVARE
Deoarece, de regula, exista diferente importante intre genotipuri din punct de vedere al cresterii calusului  chiar in absenta factorului de stres, se va urmari identificarea genotipurilor de interes agronomic care sa fie pretabile la aplicarea unor metode de selectie in vitro (capacitate ridicata de calusare si potential embriogenic ridicat). - Se va stabili in ce masura aplicarea agentului selectiv in mediul de cultura evidentiaza diferente in comportarea calusului in vitro, intre genotipuri de triticale si lucerna reprezentative pentru variabilitatea genetica existenta in cadrul germoplasmei de ameliorare la aceste culturi.- Se va  stabili daca variabilitatea existenta din punct de vedere al rezistentei patogenilor Fusarium graminearum si Fusarium oxysporum in germoplasama reprezentativa poate fi evidentiata sau nu la nivel celular, in vitro.- Vor fi  identificate mutante tolerante/rezistente de triticale si lucerna fata de patogenul Fusarium, prin utilizarea embriogenezei somatice si embrioculturii (cea de a doua metoda se va folosi in vederea obtinerii generatiei R1 si a testarii in vivo, in conditii controlate). - Se vor efectua studii cu privire la influenta micotoxinei asupra celulelor vegetale, asupra fecunditatii, starii de gestatie, mortalitatii embrionare si fetale la animale, asupra viabilitatii si greutatii produsilor la animalele hranite cu furaje infestate cu Fusarium sp. - Se vor efectua studii electronomicroscopice, determinari ale unor parametri fiziologici implicati in reactia la stres  a plantelor si animalelor. - Pe baza caracterizarii plantelor obtinute in vitro in prezenta agentului selectiv, a plantelor infestate in vivo cu toxina patogenului si a rezultatelor obtinute pe intregul lant  planta – animal - produse alimentare, vor fi elaborate metode de identificare a toxinelor patogenilor respectivi. - Se va studia timpul de metabolizare a acidului fusaric la nivel de planta, produs animalier, organism, si se va studia posibilitatea producerii de anticorpi monoclonali pentru detectarea infectiilor cu Fusarium oxysporum si Fusarium graminearum.

REZULTATE / BENEFICII ŞI SCHEMA/ PLANUL DE VALORIFICARE/ DISEMINARE

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CEEX-AGRAL 74

Advanced studies regarding the possibilities to prevent diseases produced by some toxigenic Fusarium sp. in plant-animal-human trophic chain

Financing source: State budget – Ministry of Education and Research
Program ”Cercetare de excelenta”

Contractor: National Agricultural Research & Development Institute – Fundulea

Project type: Modul 1- Complex R&D Projects, P-CD

Contract value: 1.500.000 lei 

Contract duration: 2 Years (October. 2006 – julie. 2008)

Project coordinator: NATIONAL AGRICULTURAL RESEARCH & DEVELOPMENT INSTITUTE – FUNDULEA,  N.Titulescu 1, 915200, Fundulea , tel. +40213154040, fax. +40242642044,
Project Director: Dr. Eng.Florentina Răducanu; tina@ricic.ro

Partners:
Partner 1. RESEARCH AND DEVELOPMENT INSTITUTE FOR SHEEP AND GOAT PALAS- CONSTANTA, IC BRATIANU, 248, 900316 CONSTANTA; Tel/fax:0241548186/0241639506, e-mail: stela@canals.ro, www.icdoc.ro
DIRECTOR: Dr. Dorina Nadolu

Partner 2. CONSTANTA OVIDIUS UNIVERSITY; B-dul Mamaia, nr.124,tel/fax; 024169040/02416639506; e-mail:rectorat@univ-ovidius.ro ;www.univ-ovidius.ro
DIRECTOR: Prof.univ.dr. Stela Zamfirescu


Short presentation of the project
 Thematic area :   2.    Food, agriculure and biotechologies
                                2.2  Food, health and well being; Food and nourishment from consumer standpoint, society and healt protection, inclusively link from behavior sciences; innovatory sciences concerning food and its proccesing (inclusively functional food), quality and safety chimical and mycrobiological improving of food and drinks, food chain integrity, total food chain, inclusively its transability.
The research performed by this project has as aim firstly the obtainment of some resistant somaclonal mutants of Triticale (Triticosecale Wittmack) and alfalfa (Medicago sativa) against toxigenic pathogens Fusarium oxysporum and Fusarium graminearum, by using non-conventional methods and techniques. In this respect, we have in view the improvement, in relatively short time, of quality and chemical and microbiological safe of fodders with implications on whole food chain and its trasability. Paralerly, it were performed some studies with a view to emphasize the modifications produced by pathogen toxin at bio-chemical, hematological and ultrastructural level in rats fed with milk and meat from goats and sheep exclusively fed with infected fodders (triticale and alfalfa), to produce monoclonal anticorps to detect infections with Fusarium sp. in human beings. Thus, in the first stage, were performed studies regarding some compounds of defensive mechanisms of some triticale and alfalfa genotypes (presence of chitinase in roots and stems of plants infected in vivo with F. oxysporum filtrate under controlled conditions), to determine the resistance real degree of genotypes under study compared with their phenotypical reaction. Concomitantly, testings to select genotypes, as pretability to in vitro culture were performed. The genotypes with very good answer (callusing and regeneration ability) and with superior agronomical to both in vitro studies and forage crop establishment which must ensure feeding of a goat and sheep sample during experiment were used.. The selected genotypes were introduced in a testing and selection in vitro cycle, in presence of mycotoxin culture media such as deoxynivalenol (DON) and fusaric acid (AF). Were performed profound studies regarding: i) micotoxin influence on vegetable cells tested in vitro in presence of fusaric acid (determinations of chlorophyll content, foliar area, proline content, izoenzymes specific to starting genotypes and those obtained in vitro under influence of stress induced by presence of mycotoxins into culture media and culture filtrates). ii) elaboration of some paraclinical methods to establish toxin level into serum, determinations of chemical composition of milk and meat from goats and sheep fed with infected fodders. Taking into account the toxicity of micotoxins present into culture filtrate, the treatment application were performed at daily feeding rate/fed animal, under controlled conditions, based on all measures of labour protection; iii) Were also performed lab testings on foods, from the beginning to the end of the project, in order to exactly register bio-chemical modifications in both dairy products, at the level of fed animals, and at the level of rats fed with milk and its derivates and will be performed electronomicroscopic studies at the level of some metabolic parameters involved into infection with tested mycotoxins.
The results are: obtainment, in relatively short time, of 15 triticale and 32 alfalfa cultivars with increased resistance to Fusarium graminearum and Fusarium oxysporum attack, which will ensure qualitatively fodder obtaining, free of toxins (plants for future), and on the other hand, good scientific results regarding the isolation of mycotoxins and limitation of their influence on whole trophic chain: infected plant-animal fed with infected fodder –animal fed with milk and its derivates from rats. Aspects regarding the metabolization of fusaric acid at plant level, animal product and rat organism will be elucidated with a view to produce monoclonal antibodies to detect infections with Fusarium sp. in human beings.

 

GENERAL OBJECTIVES

  1. Inducing the somaclonal variability by indirect somatic embryogenesis in triticale and alfalfa cultivars with superior agronomical traits.
  2. Elaboration of some selection and testing methodologies, with high output of regeneration ability (deficitary parameter at in vitro cultures)
  3. Identification of some efficient methods to obtain toxigenic isolates and filtrates of Fusarium sp.
  4. Identification of modification occur in regenerated plants on media supplemented with Fusarium sp. filtrates and/or DON and AF mycotoxins.
  5. Obtainment of somaclones tolerant/resistant to Fusarium graminearum and Fusarium oxysporum.
  6. Identification of bio-chemical, hematological and ultrastructural modifications at animals fed with fodders infected with Fusarium sp.
  7. Elaboration of a paraclinical method to establish toxin level.
  8. Identification of modifications produced by mycotoxins of Fusarium sp. on chemical composition of foods.
  9. Identification of mycotoxin effects in Fusarium sp. on reproduction.
  10. Elaboration of imunological test for intoxication diagnosis at humans.

OBJECTIVE FOR 2006

  1. Elaboration of some testing and selection methodologies, with high output in regeneration ability (deficitary parameter at in vitro cultures).
  2. Elaboration of an efficient method to obtain Fusarium graminearum and Fusarium oxysporum isolates, to obtain necessary filtrates for fodder treatment and supplementing of culture media. Obtainment of R0 generation.
  3. Determination of bio-chemical profile by spectophotometric method at rat blood.
  4. Organization and acquisition of animals and endowment of experimental sample.
  5. Forage crop establishment.
  6. Realization of animal feeding with ratios treated with mycotoxins; identification of modifications induced by mycotoxins of Fusarium sp. on chemical composition of foods.
  7. Dissemination of results (website, folders, symposium, conferences, workshops).

OBJECTIVE FOR 2007

  1. Obtainment of R1 triticale and alfalfa generation, resistant to mycotoxins of Fusarium sp.
  2. Identification of modifications occured in regenerated plants on media supplemented with Fusarium filtrates and/or mycotoxins DON and fusaric acid.
  3. Identification of some physiological modifications at animals fed with products infected with Fusarium sp.
  4. Identification of Fusarium mycotoxins effects on reproduction.
  5. Elaboration of a paraclinical method to establish toxin level into body of animals fed with infected milk and meat.

OBJECTIVE FOR 2008

  1. Elaboration of an experimental complex schedule based on conventional and non-conventional selection method of toxigenic Fusarium sp. isolates and resistant fodder plants (website, CD, folders, symposium, conferences, workshops).
  2. Elaboration of immunological test to diagnosis mycotoxin infections in humans. Result dissemination will take place by website, folders, CD, symposium, workshops).

 

NARDI FUNDULEA
 The identification of 20 agronomical interesting genotypes with pretability at in vitro culture, selection methods (hight callusing capacity and embriogenic capacity) were performed.
- Established in which measure the stress factor application in culture media evidentiate some difference in vitro reaction of the alfalfa and triticale genotypes, representative for genetics variability in triticale and alfalfa breeding program.
- Identification of some tolerant/resistance  Fusarium mutants, by using the somatic embriogenesis.
- Studies about mycotoxine influence on vegetal cells, on fecundity, pregnancy state, embryo death rate, on viability and weight of products at fed animals with infected fodders, will be performed.
- Based on in vitro characterization of plants obtained in presence of selective agent, on in vivo infected plants with pathogen toxine and the results obtained during the all food chain, some methods for identification of pathogen toxin will be elaborated.
- The metabolize time at plant level, food products, organism, and also the possibility to produce monoclone antibody for detection of Fusarium infection, will be studied.
- Significant reducing, by using the biotechnologie methods, of time, space, work force and the expences, coparing with those effectued in the field.

PARTENERS

The most important mycotoxins are Ochratoxin A., Patulin, Trichothecenes, Fumonisins, Zearalenone, Moniliformis, Beauvericin and Aflatoxins. The separation and detection of mycotoxins is accomplished by HPLC (high performance liquid chromatography), TLC (thin layer chromatography), GC (gas chromatography), MECC (micellar electrokinetic capillary chromatography) and ELISA.
In order to establish the pathogenity of Fusarium mycotoxins, researches were done on lactating goats who were given a solution containing fusaric acid in known concentration of 0.01 mg%, administered per os and an extract of mycotoxins in which deoxynivalenol and zearalenone were identified (41.54 µg/kg deoxynivalenol and 57.38 µg/kg zearalenone) administered per os and subcutaneous. Thus, a deterioration of the health was observed in the goat which had been given 0.01 mg% fusaric acid, with morphoclinical aspects of dehydration, nose secretions, pale mucous membranes, lack of elasticity in the skin, hair loss, diminished body weight (40% compared to the initial one). Also, the hematological profile was established (there were observed modifications of the erythrocyte and leukocyte series, which indicates anemia) as well as the biochemical profile (the rise of values for enzymes specific to the liver function AST, ALT and FA, as well as the modification of the De Ritis ratio, indicate a state of toxicosis). The morphopathological exam indicated meningeal micro-hemorrhages, splenomegaly with hemorrhagic infiltration, the distention of the gall bladder and bile retention. 
In what regards the effect of the extract obtained from wheat contaminated with 41.54 µg/kg deoxynivalenol and 57.38 µg/kg zearalenone, the observation was that the subcutaneous administration of the mycotoxin does not induce the state of toxemia (hematologically, biochemically or clinically), while the administration per os of the mycotoxin extract leads to: a drop in the leukocytes in the experimental samples, correlated with the reduction of lymphocyte and monocyte percentage, the rise in neutrophil and eosinophil percentage compared to the witness samples; the reduction of erythrocytes and of the blood plaquette volume; leucopeny associated with erythropeny and the reduction of the blood plaquette volume are signs of a mycotic intoxication. In conclusion, the oral administration (per os) has more obvious symptomatic consequences. The gastro-intestinal absorption of the mycotoxins has a stronger impact on the body’s reactivity, while the subcutaneous conjunctive tissue diminished by local reaction the crossing of the toxin into the systemic circuit. Even though there were no specific symptoms, the body’s reaction was combined “in vivo” with the hematological and biochemical blood exam, which supported the clinical observations and which proved self-evident in what regards the toxigenity of mycotoxins.
In order to establish the effect of the Fusarium toxins on reproduction and semen indexes, respectively, the contaminated wheat was administered in different doses. The semen indexes (mobility, viability after eosin-negrosin, survival indexes) displayed a reduction tendency in the samples collected at the end of the treatment, compared to the ones before its beginning; the anomaly percentage of the sperm cells rose considerably, the most affected segment being the tail (23%).
In what regards the influence of mycotoxins on the biochemical parameters of milk, there were no variations between the witness and experimental groups in goats and sheep, except the fat percentage, which registered a slight drop. The results demonstrate that even though there are many toxic trichothecenes, they are dangerous for the organism only when they are absorbed in high quantities or over long periods of time. Also, the transformation of trichothecenes in the digestive tract, especially in the rumen of ruminants, can modulate the qualitative and quantitative level of absorption and must be taken into account.
The DON and ZEA concentration dose was accomplished by HPLC and ELISA. 
The ELISA method to determine the mycotoxins in serum is simple and quick. The disadvantage of this method is generated by the interference of the glycolized products and of the metabolism products of mycotoxins, which makes this method semi-quantitative.
The absence of deoxynivalenol from the serum samples means that for the ingested DON concentration, it is either eliminated fast or transformed into other metabolism products.

Published papers:

Busuricu Florica, Zamfirescu Stela: The preliminary study of the toxicicity with Fusarium spp.at goat. Proceeding  of International Conference of the SRBBM, Timisoara 6-8 sept 2007,16 (2): 135-140, ISSN 1224-9513.

Zamfirescu Stela; Radu Marius; Schiopu Stelian: Cercetari privind influienta consumului de lucerna si triticale infestate cu Fusarium spp.asupra reproductiei la sobolani. Analele Facultatii de Stiinte ale Naturii si de Stiinte Agricole, Universitatea Ovidius Constanta. 2008, 22, (in print).

Topoleanu Irina; Zamfirescu Stela; Nadolu Dorina, Anghel Andreea: Efectul intoxicatiei acute La ovine si caprine dupa consumul de lucerna infestata cu ZEA si DON. Analele Facultatii de Stiinte ale Naturii si de Stiinte Agricole, Universitatea Ovidius Constanta, 2008, 22,  (in print).

Anghel Andreea; Coprean Dragomir; Sogorescu Elena: Studiul preliminary privind intoxicatia si transferul deoxinivalenolului din furaje in ser la ovine. A 26-ses. St. SNBC, iunie 2008, Cluj Napoca, ISSN1584-5532.


Zamfirescu Stela; Anghel Andreea; Ghita Simona; Sogorescu Elena: The quality of goat products: models and tools for evaluation and promotion. Bella-Italia, 22-26 mai 2007.

Zamfirescu Stela; Anghel Andreea;  Nadolu Dorina: „Determinarea micotoxinelor din diferite produse  de carne si lapte de la ovine si caprine”, paper communicated (poster),  ISBN 978-973-644-662-7.

Zamfirescu Stela; Nadolu Dorina: The morphostructural and physiological changes in rats and kids after acute intoxication with fusaric acid. Paper communicated (poster), International Simpozion of Nutrition on Sheep and Goats: “Nutritional and foraging ecology of sheep and goats”, Thessaloniky –Grecia 13-15 octombrie 2007.

Zamfirescu Stela; Topoleanu Irina; Nadolu Dorina: Observation concerning hematological profile in goat after acute intoxication with fusaric acid. 6 Séminaire International; Réseau FAO-CIHEAM sur les Ovins et les Caprins, Ponte de Lima Portugalia, 12-15 oct 2007.

Florentina Raducanu; Stela Zamfirescu: IMPLICATIILE MICOTOXINELOR IN LANTUL TROFIC PLANTE FURAJERE-ANIMAL -OM. Editura EX PONTO, 2008 ISBN:978-973-644-786-0.