The aim of the present study was to determine and compare the antibody (Ab) response against Fasciola gigantica in experimentally infected rabbits treated with mirazid or carnosine by ELISA. Six groups of five rabbits each, were examined (negative control, negative treated with carnosine, negative treated with mirazid, infected positive control, infected treated with carnosine and infected treated with mirazid). Each insulted rabbit was infected with 20 Fasciola metacercariae. Anti-Fasciola IgG was weekly evaluated by ELISA, in each animal serum, along the period of experiment (14 weeks). After scarifying the all animals, each liver slices were examined for the determination of flukes burden. Results showed highly significant difference between the numbers of Fasciola worms in liver of infected treated and control positive rabbits. A reduction of 54.5% in worm burden was detected in the carnosine treated group. While, the results of mirazid treated group presented complete eradication of flukes. IgG response in experimentally infected and treated groups, either with carnosine or mirazid, was significantly higher than that of control infected group. Carnosine infected group showed the highest Ab response of all groups. However carnosine action was not as potent as mirazid although it raised the general defence of the animal (Ab) plus moderate eradication effect on the worm. Thus, a combination of both carnosine and mirazid might only be recommended in patients presenting other causes of diminished immunity in addition to Fasciola infection.
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Tropical fasciolosis, caused by Fasciola gigantica, is the most economically important helminth infection of ruminants in Asia and Africa (Spithill et al., 1997). In adult cattle, the course of the disease is often between chronic to subclinical, whereas it is reported to be acute often with a high rate of mortality in young calves (Soulsby, 1982). Parasitological diagnosis of fasciolosis is, however, possible only after 13-14 weeks post-infection by demonstrating fluke eggs in feaces. By this time, major damages to the host hepatic system may have already occurred. Fasciola species represents a recognized unsolved agricultural problem responsible for marked world-wide economic losses that mounted to US$ 3 billion per year (Boray, 1985), as well as causing a not insignificant number of human infections. The estimated number of people infected with fascioliosis is 2.4 million in 61 countries, including Egypt (Motawea et al., 2001). The population at risk is 27.7 million and the number infected is at least 830, 000 (Haridy et al., 2002). The flukicide triclabendazole is the most effective drug to control Fasciola (Suhardono et al., 1991). However, the cost of treatment and the emergence of resistance to drug in sheep infected with F. hepatica suggest a need to develop more sustainable strategies.
Myrrh and loeo gum resin were obtained from the stem of Commiphora molmol (family: Burseraceae). A tree that grows in northeast Africa and the Arabian Peninsula. The council of Europe included myrrh in the list of plants that is acceptable for use in foods (Council of Europe, 1981). Purified extract of myrrh from C. molmol tree (Mirazid ®), a new herbal schistosomicidal and fasciolicidal drug has been licensed in Egypt in March, 2002. It was documented by many studies to be efficient and safe (Massoud, 1999a, b; Massoud et al., 1998; El-Gohary et al., 1999; Badria et al., 2001; Gaballah et al., 2001; Massoud et al., 2001a, b; Motawea et al., 2001; Shier et al., 2001; Hegab and Hassan, 2003). It was also proved to act as a potent larvicide (Massoud and Labib, 2003). Further, it had been proved to be effective in the treatment of dicrocoeliasis in man and animals (Massoud et al., 2003).
Carnosine, a natural dipeptide found in animal tissues, was reported to act as a beneficial regulator of many biological processes, intracellular calcium regulation, antioxidant, buffer action (Quinn et al., 1992), protecting against protein modifications (Hipkiss and Brownson, 2000). The dipeptide was also proved to have anti-inflammatory and anti-Schistosoma mansoni actions (Soliman et al., 2001) and modulating action on the immune response (Nagai and Suda, 1988; Silaeva et al., 1992; Suzuki et al., 2001). Soliman et al. (2002) showed the possible antigenic efficacy of carnosine against the individual antigens of the three different stages of S. mansoni in immunized rabbits.
Although host immune responses induced with helminthes parasites have been extensively studied yet, there is limited information on those to F. hepatica. Serological diagnosis is preferred since anti-fasciola Ab can be detected as early as 2 weeks post infection. This can facilitate early chemotherapeutic intervention (Hillyer, 1998). Immuno-diagnosis of parasitic diseases is mainly based on antibody detection (Fagbemi and Obarisiagbon, 1990). It could reveal both recent and current infections with early diagnosis. To obtain a reliable diagnostic method, many authors prepared crude antigens from whole worm (Hillyer et al., 1987; Abdel-Rahman and Abdel-Megeed, 2000). Excretory/secretory products (Espino and Finaly, 1994; Ralston and Heath, 1995) have been also prepared. The serodiagnostic methods for the detection of antibodies are quite sensitive to detect the infection in the early stages and have been exploited for the diagnosis of fasciolosis (Zimmermann et al., 1985; Sanitago et al., 1986). Adult somatic extracts and Excretory-Secretory (ES) products of F. gigantica have been used in ELISA (Maleewong et al., 1996, 1999; Youssef and Mansour, 1991) for serodiagnosis of human fasciolosis.
In the present study, the immune responses of the Fasciola infected rabbits treated with either Mirazid or carnosine were evaluated by using ELISA.
MATERIALS AND METHODS
Thirty male New Zealand white rabbits, each weighing approximately 2 kg, were obtained from Animal House, Research Institute of Ophthalmology, Cairo, Egypt. They were routinely examined for intestinal helminthes and ectoparasites.
F. gigantica metacercariae were obtained from Teodore Bilharz Schistosomal Biological Supply Program (SBSP) located in Cairo for experimental infection of rabbits. Adult worms, identified as F. gigantica based on criteria described by Sahba et al. (1972), were obtained from infected bovine livers collected from local abattoirs for preparation of antigen.
Thirty rabbits were divided into six groups of 5 animals per group.
|G1||=||Non-infected non-treated control|
|G2||=||Non-infected treated with carnosine|
|G3||=||Non-infected treated with mirazid|
|G4||=||Fasciola infected non-treated|
|G5||=||Fasciola infected and treated with carnosine|
|G6||=||Fasciola infected and treated with mirazid|
|•||Carnosine is a natural dipeptide found in animal tissues was orally injected in a dose of 100 mg day-1 for 10 days for each rabbit.|
|•||Mirazid ®), a new herbal fasciolicidal drug has been licensed in Egypt was orally given in a dose of 20 mg day-1 for six days for each rabbit.|
|•||Infected rabbits received oral 20 viable F. gigantica metacercariae at the same time of treatment.|
All rabbits were necropsied at 14 weeks after infection (end of experiment) for the determination of fluke burden. A general flow chart of all groups of animals and their treatment regime is seen in Fig. 1.
Samples Collection of Blood, Serum and Faeces
Individual blood samples were collected from all six groups at weekly intervals, up to 14 weeks. After separation of sera 0.01% merthiolate was added and stored at -20°C until use.
Coprological examination was carried out micro-scopically from 7th week post-infection, until end of the experiment by sedimentation method (Soulsby, 1965).
Preparations of Fasciola Somatic Antigen
Whole worm extract was prepared in Phosphate Buffer Saline (PBS) pH 7.2 as described by Oldham (1983). After 4-5 times washing of the live adult flukes in physiological saline, the flukes were homogenized (2 flukes mL-1) using a tissue homogenizer. The homogenate was then subjected to ultrasonication at maximum amplitude (peak to peak) for 10 min, two times, with an interval of 1 min using an Ultrasonicator (Misonix, USA). The preparation was centrifuged at 12,000 rpm for 45 min at 4°C. The supernatant was collected as somatic antigen of F. gigantica and its protein concentration was measured as described by Lowry et al. (1951).
All the pervious mentioned anti-sera were conducted using standard ELISA protocol (Zimmermann et al., 1985). Each well was coated with 10 μg mL-1 of antigen. The sera were applied at 1:100 dilution in phosphate buffer saline.
|Fig. 1:||Flow chart outlining by weeks the treatment and infection schedules|
Anti-rabbit peroxidase 1:1000 (Sigma) and ABTS were applied as conjugate and substrate, respectively. The absorbance values were recorded at 405 nm. The cut off value calculated as double fold of O.D mean negative sera.
After 14 weeks, the rabbits were sacrificed and the livers were dissected for the recovery of flukes (Wood et al., 1995). The livers were then cut into slices approximately 1 cm thick and squeezed while in the saline solution; later the liver tissue was removed and the liberated parasites were collected after 30 min of sedimentation.
Experimental data were analyzed using ANOVA (Snedecor and Cochran, 1969). Significant difference (LSD) post- hoc (SPSS computer program).
Development of the Infection
F. gigantica eggs were never seen in the faeces of infected rabbits between 9 and 14 weeks post infection (pi). The mean number of flukes recovered from infected rabbits at necropsy is shown in Table 1. Recovered flukes in the infected groups were numbered. The statistically significant results were obtained in the groups infected-treated by carnosine or mirazid. There was significant differences in the mean fluke number between groups, but the percentage of flukes was higher in the G4 (+ve control) than in the other group G5 (infected and treated with carnosine) which revealing 54.5% worm burden reduction. However, no flukes were detected in G6 (infected and treated with mirazid).
Antibody Response to F. gigantica
The levels of IgG antibody were detected in sera from all groups of rabbits by ELISA. The data were shown in Table 2 and 3 and Fig. 2. The values of the G1 (non-infected group non-treated) remained lower than the other two groups G2 and G3 which are clearly negatives throughout the experiment (Table 2, Fig. 2). The dynamics of IgG response was similar in all infected animals. In infected and treated groups G5 and G6, specific IgG levels ascended quickly and by 3 weeks (pi) showed values higher than the positive control G4 (Table 3, Fig. 2). The antibody levels of experimentally infected rabbits (G4, G5 and G6) appeared at 2 weeks post pi. There was a steady increase, reaching a peak at 8-10 weeks pi, after which antibody levels decreased and became more or less stable until 12-14 weeks pi, when levels in all groups were still clearly positive. Significantly differences could be found between groups, the highly significant values were found in G5 from the second week till the end of experiment.
In general, the reactivity of rabbit sera showed strong reactivity, highly significant values, in ELISA with Fasciola antigen in treated groups with mirazid and carnosine in comparison with non-treated.
|Table 1:||Effect of treatment of mirazid and carnosine on mean number of F. gigantica worms detected in liver of infected rabbits|
|a, b, c: Means within the same column with different letter(s) are significant (p<0.05). G4: Fasciola +ve control, G5: Infected and treated with mirazid and G6: Infected and treated with carnosine|
|Table 2:||The mean Optical Density (OD) variation in ELISA of 1/100 diluted sera of treated rabbits with mirazid or carnosine in ELISA|
|Overall mean with different letter (in rows or column) are significantly different (p<0.05)|
|Table 3:||The mean Optical Density (OD) variation in ELISA of 1/100 diluted sera of infected and treated rabbits with mirazid or carnosine in ELISA|
|Overall mean with different letter (in rows or column) are significantly different (p<0.05)|
|Fig. 2:||Dynamics of the serum antibody response in Fasciola gigantica experimentally infected rabbits, either treated with carnosine or merazid. G1: -ve control, G2: non-infected treated with carnosine, G3: non-infected treated with mirazid, G4: Fasciola +ve control, G5: infected and treated with carnosine and G6: infected and treated with mirazid|
In the present study, the statistically significant results were obtained in both infected-treated groups with either mirazid or carnosine. There was significant differences in the mean fluke number between groups, but the percentage of flukes was higher in the G4 (infected non-treated) than in the other group G5 (infected and treated with carnosine) which revealing 54.5% worm burden reduction. However, no flukes were detected in G6 (infected and treated with mirazid). Mirazid® is a new drug proved to have high therapeutic efficacy as a trematocidal drug, particularly against schistosemiasis, fascioliasis and a cestodicide (El-Gohary et al., 1999; Gaballah et al., 2001; Massoud et al., 2001a, b; Motawea et al., 2001; Shier et al., 2001; Hegab and Hassan, 2003) Carnosine, a natural dipeptide found in animal tissues, was reported to act as a beneficial regulator of many biological processes; intracellular calcium regulation, antioxidant, buffer action (Quinn et al., 1992), protecting against protein modifications (Hipkiss and Brownson, 2000). The dipeptide was also proved to have anti-inflammatory and anti S. mansoni actions (Soliman et al., 2001) and modulating action on the immune response (Nagai and Suda, 1988; Silaeva et al., 1992; Suzuki et al., 2001). These different effects of carnosine might indicate the natural selectivity of dipeptide as a universal buffer correcting many derangements (Quinn et al., 1992) for the benefit of the host. Immune suppression may be caused by parasite products as an immune evasion mechanism (Zambrano-Villa et al., 2002). Recently, O'Neill et al. (2000) identified F. hepatica excretory/secretory products (F. hepatica cathepsin L proteinase), that was able to suppress interferon gamma production. F. hepatica has other immune evasion mechanisms such as rapid turnover of the glycocalyx and cleavage of the surface-bound immunoglobulins by secreted proteases that prevent the antibody-dependent cellular cytotoxicity. The parasite immune evasion mechanisms may allow chronic infection by preventing immune clearance of the parasite (Hawn and Jong, 2001; Dalton et al., 2003).
The decreased parasite burden with treatment by carnosine explains the recovery of immune suppression in this study. This was documented by recent researches (Nutman, 2001; Pit et al., 2001). In addition, carnosine modulating the immune response could find support in many previous researches. Carnosine had the ability to bind to macrophage and lymphocyte receptors stimulating their synthetic and secretory abilities, which may eventually lead to the activation of the natural systems of body immune resistance (Nagai and Suda, 1988; Silaeva et al., 1992). It induced liberation of immune-modulator intermediates; cytokine and interlukin (Shimida et al., 1999; Suzuki et al., 2001). Carnosine modulation of immune response and decreasing apoptosis could, in part, be due to its antioxidant property (El-Moduem et al., 2002). Carnosine scavenges Reactive Oxygen Species (ROS) and prevents accumulation of already formed peroxides (Salganik et al., 2001; Boldyrev et al., 2004). It also, facilitates the removal of deleterious proteins inactivated by carbonyl groups by forming protein-carbonyl-carnosine adduct (Hipkiss and Brownson, 2000; Hipkiss et al., 2002). Further, carnosine intracellular defense mechanisms against Reactive Oxygen Species (ROS) was proposed to protect energy production and biochemical machineries of the cells (Soliman et al., 2001). The authors proved normalization of adenylate nucleoatides and adenylate energy charge. However carnosine action was not as potent as mirazid although it raised the general defense of the animal (Ab) plus moderate eradication effect on the worm. This matched the reported moderate healing ability of carnosine in other parasitic infection namely, Schistosoma mansoni. There was moderate anti-granuloma action, decrease in size of liver granuloma, diminution of its fibrotic and collagen content. However, enhancement of host immune response was shown by increased histostic and lymphocytic content (Soliman et al., 2002).
Fasciola-ELISA test is widely applicable test sensitive for acute fascioliosis and its assessment of cure (Mousa, 2001). In the present study, the Ab levels of experimentally infected rabbits appeared at 2 weeks pi. There was a steady increase, reaching a peak at weeks 8-10 pi, after which Ab became more or less stable until 12-14 weeks pi. Significantly differences could be found between groups, significant higher values were found in G5 from the second week till the end of experiment. These data were in agreement with other previous researches (Kendall et al., 1978; Zimmermann et al., 1982; Mousa, 1994; Mbuh and Fagbemi, 1996), when antigens derived from adult worms were used. These findings were also in agreement with Massoud et al. (2004) who detected the mean value of Indirect Heamagglutination Assay (IHA) titer was 966 before treatment that decreased significantly after therapy in 88.6% of cases were still having high titers. Massoud et al. (2001b) reported marked decline of Ab three months after mirazid therapy using indirect fluorescent antibody test. In comparison with other fasciolicidal drugs, 90% of patients treated with praziquantel or bithionol had high antibody titer 3 months after treatment (Espino et al., 1992) and 50% of those treated with triclabendazole and height titer 3 months after treatment (Hammouda et al., 1995).
It is concluded that Mirazid® is, up till now, still the most safe and effective fasciolicidal drug at the clinical, parasitological and immunological levels. Although carnosine proved to be a save and effective immunostimulant. These data inspires more hope for further research on prophylactic and therapeutic potencies of carnosine on F. gigantica infection, one of cosmopolitan human insults. Yet, alone it did not eradicate fasciola infection. Hence carnosine could be given in conditions associated with immune suppression. Another proposal is to further search treatment of different doses of carnosine combined with mirazid.
- Abdel-Rahman, E.H. and K.N. Abdel-Megeed, 2000. Molecular identity of major cross-reactive adult antigens in Fasciola gigantica, Toxocara vitulorum and Moniezia expansa. J. Egypt. Soc. Parasitol., 30: 561-571.
- Badria, F., G. Abou-Mohamed, A. El-Muwafy, A. Massoud and O. Salama, 2001. Mirazid: A new schistosomicidal drug. Pharmacol. Biol., 39: 127-131.
- Boldyrev, A.A., E. Bultgina, T. Leinsoo, I. Petrushanko, S. Tsubone and H. Abe, 2004. Protection of neuronal cells against reactive oxygen species by carnosine and related compounds. Biochem. Mol. Biol., 137: 81-88.
- Dalton, J.P., S.O. Neill, D. Stack, P. Collins and M. Sakiyo et al., 2003. Fasciola hepatica Cathepsin L. like roteases: Biologic, function and potential in development of firstgeneration liver fluke vaccines. Int. J. Parasitol., 33: 1173-1181.
- El-Moduem, M., G. Laurent, M.P. Mingeot-Leclercq, H.S. Taper, J. Cumps and P.M. Tulkens, 2000. Apoptosis in renal proximal tubules of rats treated with low doses of aminoglycosides. Antimicrob. Agents Chemother., 44: 665-675.
- Espino, A.M. and C.M. Finaly, 1994. Sandwich enzyme-linked immunosorbent assay for the detection of excretory Secretory antigensin humans with fascioliasis. J.Clin. Mirobiol., 32: 190-193.
- Fagbemi, B.O. and I.O. Obarisiagbon, 1990. Comparative evaluation of the enzyme linked immunosorbent assay in the diagnosis of natural Fasciola gigantica infection in cattle. Vet. Q., 12: 35-38.
- Hammouda, N.A., S.T. El-Mansoury, M.Z. El-Assouni and Y. El-Gohary, 1995. Therapeutic effect of ticlabendazole in patients with fascioliasis in Egypt: A preliminary study. J. Egypt. Soc. Parasitol., 25: 137-143.
- Haridy, F.M., T.A. Morsy and N.I. Gawish, 2000. The potential reservoir role of donkeys and horses in zoonotic fascioliasis in Gharbia G. Egypt. J. Egypt. Soc. Parasitol., 32: 561-570.
- Hegab, M.H.A. and R.M. Hassan, 2003. Role of circulating fasciola antigens and IGG4 isotype in assessment of cure from fascioliasis. J. Egypt. Soc. Parasitol., 33: 561-570.
- Hillyer, G.V., E.T.M. Haroun, A. Hernandez and M.S.D. Galanes, 1987. Acquired resistance to Fasciola hepatica in cattle using apurified adult wormantigen. Am. J. Trop. Med. Hyg., 37: 363-369.
- Hipkiss, A.R. and C. Brownson, 2000. Carnosine reacts with protein carbonyl groups: Another possible role for anti-ageing peptide? Biogerontology, 1: 217-223.
- Hipkiss, A.R., C. Brownson, F. Mariana, E. Ruiz and A. Ferro, 2002. Reaction of carnosine with aged protein: Another protective process? Ann. New York Acad., 959: 285-294.
- Kendall, S.B., I.J. Sinclair, G. Everet and J.W. Parfitt, 1978. Resistance to F. hepatica in cattle. I. Parasitological and serological observations. J. Comp. Pathol., 88: 115-122.
- Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275.
- Massoud, A., S. El Sisi, O. Salama and A. Massoud, 2001. Preliminary study of therapeutic efficacy of a new fasciolicidal drug derived from Commiphora molmol (Myrrh). Am. J. Trop. Med. Hyg., 65: 96-99.
- Massoud, A., T.A. Morsy and F.M. Haridy, 2003. Treatment of dicrocoeliasis in man and animals with Mirazid. J. Egypt. Soc. Parasitol., 33: 437-442.
- Massoud, A.M. and I.M. Labib, 2000. Larvicidal activity of Commiphora molmol against Culex pipiens and Aedes caspius larvae. J. Egypt. Soc. Parasitol., 30: 101-115.
- Massoud, A.M., N.M. El-Kholy, F.A. El-Shennawy and R.E. Farag, 2004. Study of some immune aspects in patients with fascioliasis before and after Chommiphora molmol (Mirazid) treatment. J. Egypt. Soc. Parasitol., 34: 315-332.
- Mousa, W.M., 2001. Evaluation of cercarial antigen for serodiagnosis of fascioliasis in experimentally and naturally infected sheep. Vet. Parasitol., 97: 47-54.
- Nutman, T.B. and V. Kumaraswami, 2001. Regulation of the immune response in lymphatic filariasis: Perspectives on acute and chronic infection with Wuchereria bancrofti in South India. Parasite Immunol., 28: 389-399.
- O'Neill, S.M., M.T. Brady, J.J. Callanan, G. Mulcahy, P. Joyce, K.H. Mills and J.P. Dalton, 2000. Fasciola hepatica infection down regulates the immune responses in mice. Parasitol. Immunol., 22: 147-155.
- Quinn, P.J., A.A. Boldyrevt, V.E. Formazuyk, 1992. Carnosine: Its properties, functions and potential therapeutic applications. Mol. Aspects Med., 13: 379-444.
- Shier, Z., A.A. Nasr, A. Massoud, O. Salama, G.A. Badra, H. El-Shennawy and S.M. Hannad, 2001. A safe effective herbal antischistosomal therapy derived from myrrh. Am. J. Trop. Med. Hyd., 65: 700-707.
- Soliman, K.M., A.K. El-Anasry and A.M. Mohamed, 2001. Effect of carnosine administration on metabolic parameters in bilhariza-infected hamsters. Comp. Bicohem. Physiol., 129: 157-164.
- Soliman, K.M., M. Abdel Aziz, Y.H. Nassar, S. Abdel-Sattar and A.K. El-Ansary, 2002. Effect of carnosine on bilharzial infestation, biochemical and histochemical studies. Comp. Biochem. Physiol., 131: 535-542.
- Spithill, T.W., D. Piedrafita and P.M. Smooker, 1997. Immunological approaches for the control of fasciolosis. Int. J. Parasitol., 27: 1221-1235.
- Wood, I.B., N.K. Amaral, K. Bairden, J.L. Duncan and T. Kassai et al., 1995. World association for the advancement of veterinary parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Vet. Parasitol., 58: 181-213.
- Zambrano-Villa, S., D. Rosales-Borjas and J.C. Carrero, 2002. How protozoan parasites evade the immune response. Trends Parasitol., 18: 272-278.