Publications

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2018
Osman, A. H., E. M. M. F. Hala, A. M. Zakia, A. M. Mahmoud, and et al, "RABBIT'S HEALTH AND ENVIRONMENTAL IMPACTS OF EXPLOITATION TREATED BIODEGRADABLE POLLUTION WATER BY JOHKASOU SYSTEM", Taiwan Veterinary Journal, vol. 44, issue 1, pp. 1-8, 2018.
2017
NaveenKumar, S., M. A. Hassan, M. A. Mahmoud, and A. Al-Ansari, "Betanodavirus infection in reared marine fishes along the Arabian Gulf", Aquacult Int, vol. 25, pp. 1543–1554, 2017.
Hassan, M. A., E. A. Noureldin, M. A. Mahmoud, and N. A. Fita, "Molecular identification and epizootiology of Aeromonas veronii infection among farmed Oreochromis niloticus in Eastern Province, KSA", Egyptian Journal of Aquatic Research, vol. 43, pp. 161-167, 2017.
2016
Mahmoud, M. A., M. Abdelsalam, O. A. Mahdy, H. E. M. F. Miniawy, and et al, "Infectious bacterial pathogens, parasites and pathological correlations of sewage pollution as an important threat to farmed fishes in Egypt", Environmental Pollution, vol. 219, pp. 939-948, 2016.
2015
Karima, M. F., H. M. Aziza, R. A. Hasnaa, and M. A. Mahmoud, "Inhibition of cadmium- induced genotoxicity and histopathological changes in Nile tilapia fish by Egyptian and Tunisian montmorillonite clay", Ecotoxicology and Environmental safety, vol. 119, issue 2015, pp. 140- 147, 2015.
Hassan., M. A., W. S. Soliman., M. A. Mahmoud., and M. I. Sami Shabeeb Al-Shabeeb, "Prevalence of Bacterial Infections among Cage-Cultured Marine Fishes at the Eastern Province of Saudi Arabia.", Research Journal of Pharmaceutical, Biological and Chemical Sciences , vol. 6, issue 4, pp. 1112-1126, 2015.
2014
Nisreen, E. M., A. M. Mahmoud, and M. M. Fahmy, "Parasitological and Comparative Pathological Studies on Monogenean Infestation of Cultured Sea Bream ( Sparus aurata , Spariidae) in Egypt", Oceanography, vol. 2, issue 4, pp. 129, 2014.
Hassan, M. A., H. A. M. Osman, and M. A. Mahmoud, "Studies on Dermocystidiosis (Yellow Muscle Disease) among Some Marine Fishes of Arabian Gulf and Red Sea Coast, Jeddah, Saudi Arabia", Middle-East Journal of Scientific Research, vol. 22 , issue 4, pp. 478-487, 2014.
2011
Mahmoud, A. M., and A. N. Z. Gindy, "Treatment of Monogenic Parasites in Imported Hybrid Red Tilapia Fries ( Oreochromis SPP) In the Sultanate of Oman.", Agricultural and Marine Sciences, vol. 16, pp. 67-72, 2011. Abstract

In February, 2008, 12000 fries of monosex red tilapia hybrid (Oreochromis SPP) of 0.2 g and 12000 fries of monosex Nile tilapia (Oreochromis niloticus) of 0.5 g were imported from a hatchery in South East Asia and stocked in concrete raceways at an agriculture farm in the Barka Region about 80 km from Muscat. Three days after stocking, mortality problems were observed. The preliminary inspection revealed abnormal flashing movements of the fish with body scratching against the walls. Faint grayish white discoloration was observed externally on the skin and fins with focal hemorrhagic areas. Examination of skin and gill biopsies demonstrated the presence of a large number of small sized monogenea parasitic worms attacking the tissue of these body regions. Histopathological observations revealed tissue reactions against the parasites and demonstrated a severe dermatitis of the skin, lamellar oedema and hyperplasia of the branchial tissue. A concomitant treatment regime using mebendazole, salt and formalin was practiced for three successive days and repeated three times every week together with parallel management procedures to enhance water quality during treatment. After treatment, a reduction of mortality was noticed within three days and completely ceased after one week at which time biopsy examinations revealed the absence of any parasitic agents. There were no deaths reported among the Nile tilapia, which were raised on the same farm.

2010
M. Moustafa, Laila. A. Mohamed, M. A. Mahmoud, and E. -gendyM. Y. W.S. Soliman, "Bacterial Infections Affecting Marine Fishes in Egypt", Journal of American Science , vol. 6, no.11, pp. 603-612, 2010. Abstract

Some fish species are suffering from continuous depletion due to devastating environmental changes at their native aquatic environment. Qarun Lake and Suez Gulf are among the most vulnerable areas. Thus, representative fish samples from those areas were inspected for the presence of any fish pathogenic bacteria. The inspected samples included six different species; Epinephelus tuvina, Siganus rivulatus, and Dedlechilus labiosus native to Suez Gulf at Suez governorate; Tilapia zilli, Mugil capito and Solea vulgaris native to Qarun Lake at El- Fayoum governorate. A total of 600 samples were examined throughout the different year seasons. Gram positive and negative fish pathogenic bacteria were isolated from a total of 245 fish sample. Among those samples, the following bacteria were retrieved in the following percentages respectively, 17.55% (V. anguillarum), 16.73% (V. alginolyticus), 15.51% (P. piscicida), 15.91% (Ps. fluorescens), 13.46% (S. fecalis), 11.02% (A. hydrophila), 6.12% (A. sobria) and 3.67% were infected with Staph. aureus. The Siganus rivulatus was the highest infected fish species with a prevalence of 8.33%, while Mugil capito was the lowest infected species (5.67 %). The highest total prevalence of bacterial infection was recorded in summer season (40.81%) while the lowest was recorded in winter (15.91%). The aforementioned bacterial isolates were successfully re-isolated from experimentally infected fish. The retrieved isolates were matched against standard isolates as well as confirmed to be positive using semi- automated (API 20 E) and conventional biochemical tests.

2009
Girgis, S. M., and M. A. Mahmoud, "PROTECTIVE EFFECT OF VITAMIN C AGAINST GENOTOXICITY AND PATHOLOGICAL CHANGES CAUSED BY SOME HEAVY METALS IN OREOCHROMIS NILOTICUS", J. Egyp. Vet. Med. Assoc., vol. 69, no. 4, pp. 263-285, 2009. Abstract

The present study was carried out to evaluate the protective effect of vitamin C (vit. C) against genotoxic and pathological changes induced by arsenic (As), zinc (Zn) and their mixture in Oreochromis niloticus. Fish of almost the same weight and size were divided into 13 groups (6 groups were exposed for 3 weeks and the other 6 groups were exposed to the same treatment but for 6 weeks) beside the control group (10 fish/group). The 1st group was served as control, the 2nd group was exposed to 2.9 mg/L of As in the form of sodium salt in water for 3 weeks. The 3rd group was exposed to As plus vit. C (10mg/kg diet equivalent to 30 gm/kg bw). The 4th group was exposed to 3mg/L of Zn in the form of zinc sulfate. The 5th group was exposed to Zn and vit. C. The 6th group was exposed to both As and Zn. The 7th group was exposed to As, Zn and vit. C. Five samples from each group were sacrificed at 3rd and 6th week of the experiment, respectively, and subjected to cytogenetical and histopathological examination. Gross lesions were recorded along the time of exposure. The results revealed genotoxicity in O.niloticus exposed to As and/ or Zn for 6 weeks. There was a significant increase of micronuclei (Mn) in all As and/or Zn exposed fish groups, compared to the control, while the treatment with vit. C significantly decreased the frequency of Mn in all As and/or Zn exposed groups. The histopathological examination revealed severe pathological lesions in gills, skin, muscles and liver in case of As toxicity, while in case of Zn toxicity, the changes were common in liver and spleen and other internal organs. In both cases, vit. C protects the tissue against such toxicity. In conclusion, this study proved that As and Zn had genotoxic effect on O.niloticus and that appear in the significant increase in the frequency of Mn induced by these metals, whereas the treatment with vit. C reduced that genotoxic effect especially with long term treatment. In addition, the histopathological lesions were recorded in As and/or Zn exposed groups for 3 w, whereas the alterations were more clear in 6 w exposed groups. Minor or no lesions were noticed in all vit. C treated groups.

Mahmoud, M. A., S. M. Aly, A. S. Diab, and G. John, "The role of ornamental goldfish Carassius auratus in transfer of some viruses and ectoparasites to cultured fish in Egypt: comparative ultrapathological studies", African Journal of Aquatic Science , vol. 34, no.2, pp. 111–121, 2009. Abstract

Goldfish, common carp and Nile tilapia were sampled between September 2002 and May 2003 to investigate lesions induced byviral diseases and ectoparasites. Goldfish exhibited neoplasms, Dermocystidium sp., Ichthyophthirius multifiliis (Ich), Trichodinareticulata, Lernaea cyprinacea and systemic infections. Neoplastic and systemic infections in goldfish were associated with viralinfection. Oreochromis niloticus and Cyprinus carpio were mainly infested with several species of Trichodina and monogeneans.
The presence of Dermocystidium sp. in carp, and viral particles in the liver and spleen of O. niloticus and C. carpio, wasuncommon. Microscopically, intracytoplasmic inclusion bodies in the hepatic cells and lymphocytic infiltration in other internalorgans of the three fish species were seen. Ultrastructural study showed enveloped viral particles in some cells in all fish species.The recorded parasitic infections were associated with degenerative, necrotic, inflammatory and proliferative changes in the skin and underlying muscles. Imported ornamental goldfish may be a source of infection to carp and native tilapia. Histopathology andtransmission electron microscopy demonstrated the etiology and pathogenesis of infection and the preliminary diagnosis of viralinfection, which is not common in Egypt. It is recommended that goldfish be excluded from edible-fish aquaculture ponds. Strictregulations regarding the transfer and quarantine of imported fish should be implemented.

2008
Mahmoud, A. M., and A. I. Tanios, "Pathogenicity of Aeromonas hydrophila in chickens", Egyt. J. of Comp. Path. & Clinical Path., vol. 21, no. 3, pp. 88-110, 2008. Abstract

Seventeen isolates of Aeromonas hydrophila were isolated from 250 commercial broiler chicks with an incidence of 6.8 %. Most A. hydrophila isolates (88.24 %) were positive for exotoxin assay and congo red binding test, while 52.94 % were positive for crystal violet binding activity. Most strains of A. hydrophila were sensitive to chloramphenicol, ciprofloxacin and norfloxacin followed by gentamicin and neomycin while nalidixic acid, tetracycline, streptomycin and trimethoprim sulphamethoxazole had moderate effect. On the other hand, all A. hydrophila strains were resistant to amoxicillin, cephalothin, erythromycin and penicillin G. All chicks infected with 1.5 X 109 organisms via subcutaneous and yolk sac were dead within 24 h. A. hydrophila was isolated from most organs. The lesions observed included congestion in the internal organs and few cases showed hepatic and muscular petechiae. Histopathological examination of experimentally infected chicks revealed severe necrosis in hepatic, splenic and muscular tissue in the two days old chicks. The ultrastructural study of this group showed presence of the bacilli inside the hepatocytes and macrophages with marked cellular changes. An attempt was made to determine a correlation between level of exposure and mortality. It was found that the mortality rate was relatively high (52.5 %) than in lower dose (35 %). A. hydrophila was isolated from most organs examined. Marked degenerative and necrobiotic changes in both hepatic and splenic tissue and characteristic muscular lesions manifested by muscular hemorrhage, degeneration, oedema and myositis in low dose treatment group. In the group injected with high dose, the lesions were more severe and characterized by diffuse areas of necrosis in hepatic tissue, thrombus formation in the blood vessels together with large number of bacterial colonies and bacilli in the hepatic tissue. Marked muscular necrosis and myophagia were also noticed. The ultrastructural study using transmission electron microscope for this group showed heterocells and hepatocytes contain bacilli. In other cases, the bacilli were present in the phagosomes of phagocytic cells in the splenic tissue. Cytopathological lysis was common evidence in the examined cells.

2007
Mahmoud, A. M., A. E. R. H. Abeer, and A. - A. A. Nashwa, "Effect Of Confinement Stress On Behaviour, Performance, Clinicopathological And Histopathological Alterations Of Nile Tilapia Challenged With Aeromonas hydrophila With Regard To The Blue Light As Stress Inhibitor", Veterinary Medical Journal, vol. 55, no.3, pp. 687-717, 2007. Abstract

The present study was carried out to assess the effects of blue light as antistressor and its effect on susceptibility of Nile tilapia (Oreochromis niloticus) to Aeromonas hydrophila (A. hydrophila) infection. A total number of (120) O. niloticus was divided into 3 experimental groups (40 fish / group). Group 1 (G1), was exposed to normal day light and considered as control group. Group 2 (G2), was exposed to blue light. Group 3 (G3), was exposed to white light (fluorescent illumination). After 15 days of light exposure, fish in groups 2 and 3 were confined into small area and then challenged with A. hydrophila infection. The obtained data revealed that during exposure to blue light, the fish of (G2) recorded significant lower proportion in behavioural parameters including factors of fright behaviour "creeping, oblique plan position and air-gulping" than those of﴾G3﴿. On the other hand, (G2) showed lower behaviour alterations for fright behaviour during confinement and infection than those of﴾G3﴿. Contrarily, it also had significantly higher proportions of aggressive behaviour during exposure to light, confinement and infection than﴾ G3﴿. SO blue light improved behaviour and fish become more comfortable in (G2) than (G3). Moreover, fish of (G2) showed higher significant growth performance during exposure to blue light and little reduction in growth performance during exposure to both confinement and infection than those of (G3). The clinicopathological studies revealed significant increase in the stress response indicators after exposure to confinement stress in (G.3). Marked elevation in the serum cortisol, glucose levels and significant leukocytosis associated with heterophilia and lymphopenia was observed in group 3 (G3). On the other hand, non significant changes were observed in the measured stress parameters in group (G 2) which exposed to the blue light. Exposure of stressed fish to A. hydrophila infection induced significant leukocytosis with heterophilia, monocytosis and lymphopenia in both (G2&G3). Analysis of serum biochemical constituents showed significant elevation in ALT, AST, BUN, creatinine and glucose while significant reduction in the total proteins, albumen and globulin was achieved. These changes were more pronounced and early detected in A. hydrophila infected stressed fish exposed to white light compared to those exposed to blue light From pathological point of view, fish group which exposed to blue light and infection revealed early positive immune response "activation of melanomacrophage center of spleen" but with time dependent adverse pathological lesion. The lesions were noticed in spleen and gills after 2 weeks. Regeneration of the most observed lesions were detected after 4 weeks. On the other hand, the fish group exposed to white light and infection showed more advanced lesions including diffuse necrosis in hepatic tissue with additional changes in the kidneys. So, the blue light has a protective effect aganist stress in Nile tilapia.

abd elaziz, M., M. Ali, S. yassin, and A. S. raafat and rashad, "Study on the effects of some probiotics on the performance, clinicopathological and histopathological changes of Oreochromis niloticus", Veterinary Medical Journal, vol. 55, no.3, pp. 879-897, 2007. Abstract

The presen study was carried out to evaluate the use of some probiotics on the growth rate of tilapia fishes and to determine its effect on some hematological and histopathological parameters. Two different commercial probiotics which includes biogen and moreyeast were evaluated for 8weeks. In the experiment, fifty Oreochromis niloticus fishes were classified to 3 groups,Group (1): consisted of 20 fishes which fed diet supplemented by biogen at a dose of 2 kg/ton diet. Group (2): consisted of 20 fishes which fed diet supplemented by moreyeast at a dose of 5 kg/ton diet and Group (3): was control group which consisted of 10 fishes fed diet without probiotics. At the end of the experimental period both biogen and moreyeast administrated groups revealed a significant increase in the body weight gain (W.G.). The results showed significant increase in the values of RBCs, PCV and Hb throughout the experimental period except group 1 (received biogen) at the 6th week of experiment comparing to control group. Fish group received moreyeast (Group 2) had the pronounced effect than group 1. Assessment of leukogram revealed significant leukocytosis allover the experimental period in different groups except during the 2nd week in group 2 (received moreyeast) and at the 6th week in both probiotic administrated groups comparing to control group. Fish group received biogen had pronounced effect comparing to moreyeast administrated group. differential leukocytic picture was recorded. Histopathological examination revealed The biogen and moreyeast administerd groups showed marked activation of the melano-macrophage centers which is a component of reticuloendothelial system and hence part of the defensive system of fish indicating immunoenhancment. the study concluded that: The examined probiotic were safe for the Oreochromis niloticus fish.

Khalil, W. K. B., M. A. Mahmoud, M. M. Zahran, and K. F. Mahrous, "A sub-acute study of metronidazole toxicity assessed in Egyptian Tilapia zillii.", Journal of applied toxicology, vol. 27, pp. 380-390, 2007. Abstract

Metronidazole (MTZ), an antiparasitic and antibacterial compound, is one of the world’s most widely used drugs. Despite being considered as a rodent mutagen and a carcinogen, it is still widely used in humans for the treatment of infections with anaerobic organisms. Therefore, the main objective of the current study was to evaluate the in vivo toxicity of MTZ using the micronucleus (MN) assay and random amplified polymorphism DNA (RAPD-PCR) analysis as well as histopathological examination in Tilapia zillii. Moreover, the protective effect of vitamin C (VitC) against toxi- city of MTZ was investigated in the present study. Fish were treated with three doses of MTZ (5, 10 and 20 mg/ l) alone or in combination with VitC (200 mg/ kg food) at several time intervals (2 days, 7 days and 14 days). The results of the present study showed a significant effect of MTZ on micronucleus formation and changes in polymorphic band patterns as well as induction of different histopathological alterations in Tilapia zillii. The effects of the drug were reduced when fish were exposed to a combination of MTZ and VitC.

2006
Karima, F. M., K. B. Wagdy, and A. M. Mahmoud, "Assessment of toxicity and clastogenicity of sterigmatocystin in Egyptian Nile tilapia", African Journal of Biotechnology, vol. 5, no.12, pp. 1180-1189, 2006. Abstract

The increasing presence of genotoxic pollutants in the aquatic environment has led to the development of quick monitoring methods. Sterigmatocystin (Stg) is closely related to mycotoxins and has the carcinogenic potency in the experimental animal models. The exposure to genotoxic agents will give rise to alterations of DNA structure that can lead to abnormal changes of DNA fingerprints. Therefore, we have applied the random amplified polymorphism DNA (RAPD) method to evaluate the genotoxic effects of Stg and to determine if the Egyptian montmorillonite (EM) has a protective effect against Stg. The experiment was conducted in vivo to evaluate the ability of EM at a level 0.5 mg/kg body weight (bw) to prevent the toxicity and genotoxicity induced by Stg in the Nile tilapia fish. Fishes were orally administrated with EM in corn oil with or without Stg (1.6 µg/kg bw) twice a week for 4 weeks. Blood and tissue samples were collected at the end of the treatment. The results revealed that Stg had genotoxic and toxicopathological effects in Oreochromis niloticus fish. The genotoxic effects were indicated by appearance of some changes in polymorphism band patterns including lost of stable bands or occurrence of new bands. There also exists a distinct distance between the band patterns of exposed fish and protected or control fish samples. The effects on the tissues were manifested by different histopathological lesions in different organs including hyperplastic proliferation of branchial epithelium, necrobiotic changes in hepatic tissue and destruction of components of the spleen. These responses were virtually abolished or markedly decreased when fishes were exposed to EM combined with Stg. It could be conclude that addition of EM resulted in the inhibition of the toxicity and clastogenicity of Stg.

Elsayed, E. E., N. E. E. Dien, and M. A. Mahmoud, "Ichthyophthiriasis: Various Fish Susceptibility or Presence of More than one Strain of the Parasite?", Nature and Science, vol. 4, no.3, pp. 5-13, 2006. Abstract

White spot disease is one of the devastating protozoal infections affecting freshwater fish. Commonly known as “Ich”, the Ichthyophthiriasis can infect almost all freshwater fish causing devastating losses in susceptible fish. In the present study, an outbreak of Ichthyophthiriasis erupted in one of the holding tanks of two ornamental fish species, Siamese shark (Pangasius sutchi) and goldfish (Carassius auratus var.bicausatus). Initial observation of the outbreak showed that only Pangasius sutchi was affected by typical white spots associated with mortalities. However, Carassius auratus, a known susceptible species for Ichthyophthirius multifiliis (Ich) in the same aquarium showed only mild erythema that disappeared during the course of infection with no mortalities. To confirm the previous observation, an experimental designs was performed in which infection with Ichthyophthirius was induced in Pangasius sutchi species alone. Cohabitation was performed between the Ich-induced Pangasius sutchi and Carassius auratus. Three days after the induction, Pangasius sutchi started showing the typical clinical signs. Mortalities associated with severe infection were recorded in Pangasius sutchi by 7th day after infection. Associated Carassius auratus showed only mild erythema that disappeared by the end of experiment. Histopathological examination of skin from both species in natural and experimental infection was performed to evaluate the severity of infection on the tissue level. Substantial numbers of typical large size trophonts surrounded by layers of fibrous tissue, melanophores and hemorrhages were detected in dermal and epidermal layers. Underlying myodegeneration was also associated the skin lesions in Pangasius sutchi. In contrary, pathological changes in the skin of Carassius auratus were mild and few numbers of immature trophonts were noticed in the epidermal layers. Possible reasons for such infection discrepancies between the two susceptible species are discussed.

MAI, I. B. R. A. H. E. M. D., H. A. Hussien, and A. M. Mahmoud, "Spring Viraemia of Carp disease: Experimental Infection of Cultured Common Carp (Cyprinus Carpio L.) in Egypt", Veterinary Medical Journal, vol. 54, no.4, pp. 887-898, 2006. Abstract

Experimental oral infection of common carp (C. carpio L.) fish was carried out via stomach tube with 2 local positive samples of Spring Viraemia of Carp Virus (SVCV) at water temperature 14± 20C. Investigation of the pathogenesis of SVCV to common carp (C. carpio L.) revealed that virus was detected in gills at the 3rd day post infection, and in the internal organs one week post infection. Clinical and post mortem examination of experimentally infected fish were recorded. The established infection was confirmed using monoclonal antibodies against SVCV in dot ELISA technique and electron microscopy. Histopathological picture of the experimentally infected common carp (C. carpio L.) showed severe changes in gills, liver and intestine.

2005
Marzouk, M. S. M., M. M. Ali, M. Basma Shalaby, and M. I. Mahmoud, A.M. and ABD EL-Salam, "A contribution on anaerobic bacterial infection in cultured freshwater fish", J. Egypt. Vet. Med. Assoc. , vol. 65, no.3, pp. 123-140 , 2005. Abstract

In this work, the incidence and occurance of anaerobic bacterial pathogen in different freshwater fish farms have been investigated. The results indicated the presence of C. perfrengens, C. sporogenes, C. birfermentans, Actinomyces species and peptostreptococcus species in Nile tilapia ( O. niloticus), C. carpio in soil of earthen ponds using poultry droopings as organic fertilizers, while nile tilapia ( O. niloticus) collected from concrete ponds were negative for isolation of anaerobic bacteria.Typing of isolated C. perfringens indicated that the isolates were belonging to the toxogenic A and D types. Examination of water samples revealed the presence of great alterations in the physicochemical and microbiological parameters, particularly, in samples collected from earthen ponds.
The histopathological changes of experimentally inoculated fishes with toxogenic isolates of C. perfringens were fully demonstrated.

2004
A.M.Mahmoud, N. M.Ezz-Eldien, and E.E.Elsayed, "Tissue Protozoa (Myxobolus dermatobia) from the Eye of Tilapia zillii in Egypt", The First International Conference of the Veterinary Research Division, NRC, Egypt, 15-17 February , 2004.
Elsayed, E., M. A. Mahmoud, and N. E. ElDien, "Ichthiophthiriasis: Atypical outbreak In Two Suscetible Ornamental Fish Species In Egypt Under The Same Environmental Conditions", 29th ANNUAL EASTERN FISH HEALTH WORKSHOP, U.S.A, March 22-26, 2004.
Sekena, H. A. E. - A., M. A.Abdel-Wahab, M. A.M, and M. F. Azza M. Hassan, Karima, "Chemoprevention of Barley and Sage against acrylamide-Induced genotoxic, biochemical and histopathological alterations in rats", The Egyptian Journal of Hospital Medicine, vol. 15, pp. 40-56, 2004.
Sekena, A. - A. H., and A. M. Mahmoud, "Mutagenic and Histopathological Effects of Ribavirin Drug on Mice", J. Egypt. Vet.Med. Assoc., vol. 64, no.2, issue 2, pp. 153-149, 2004.
Mahmoud, A. M., "Pathological Study on Myxosporean Disease in Cultured Nile Tilapia (Oreochromis niloticus) Reared in Sandy and Muddy ponds in Egypt", Egyt. J. Comp. Path. & Clinic. Path, vol. 17, no.2, pp. 250- 261, 2004.
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