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Setaria equina: In vivo effect of diethylcarbamazine citrate on microfilariae in albino rats
Experimental Parasitology 126 (2010) 603–610
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Setaria equina: In vivo effect of diethylcarbamazine citrate on microfilariae in albino rats G.A. El-Shahawi a, M. Abdel-Latif a,*, A.H. Saad b, M. Bahgat c,** a
Department of Zoology, Faculty of Science, Beni-Suef University, Egypt Department of Zoology, Faculty of Science, Cairo University, Egypt c Therapeutic Chemistry Department, Infectious Diseases and Immunology Laboratory, The Center of Excellence for Advanced Sciences, The National Research Center, Dokki, Cairo 12311, Egypt b
a r t i c l e
i n f o
Article history: Received 29 October 2009 Received in revised form 17 May 2010 Accepted 15 June 2010 Available online 18 June 2010 Keywords: Setaria equina Albino rats Experimental infection Diethylcarbamazine citrate Organs
a b s t r a c t Although diethylcarbamazine citrate (DEC) is successful drug in eliminating human filariasis, yet, its mode of action is still debatable. Herein, the effect of DEC to treat albino rats infected with the animal filarial parasite Setaria equina was tested. Microfilarial (mf) counts and sections from liver, lung, kidney as well as spleen were investigated at different time points after treatment by light microscopy. After 45 and 300 min of treatment, a significant decrease in blood mf was observed accompanied by adherence of degenerated mf to both kupffer cells and leukocyte in liver sections. In lung sections, loss of sheath was observed at 45 min, while degeneration was observed at later time points. In kidney sections, more mf counts and less matrix were observed in the glomeruli at all time points after treatment. Degenerated mf were observed in spleen sections only at, late time point, 480 min after treatment. In conclusion, one of the possible mechanisms by which DEC reduces blood microfilarial count is trapping larvae in organs and killing them through cellular adherence. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction DEC is a piperazine derivative having both microfilaricidal and macrofilaricidal activities. The success of control programs depends on whether the adult worms are killed or temporarily sterilized and the extent of microfilarial clearance (Eberhard et al., 1997). Therapeutic efficacy of DEC was described more than 50 years ago, yet, its mode of action is still debatable (Hewitt et al., 1947; Santiago-Stevenson et al., 1947). Treating infected experimental animals with filarial parasites by DEC caused elimination of mf from circulation (Zahner et al., 1978; Weiner et al., 1986; Mukhopadhyay et al., 1996) that was referred to the highest rate of cellular adherence, phagocytosis and subsequent lysis of mf in the sinusoids and parenchymal inflammatory foci of the liver (Schardein et al., 1968; Zahner et al., 1978; Piessens and Beldekas, 1979; Fanning and Kazura, 1985). The predominant inflammatory cells in the liver of Dirofil-
* Corresponding author. Fax: +20 822334551. ** Correspondence to: M. Bahgat, Current address: Department of Infection Genetics, the Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. E-mail addresses: [email protected] (M. Abdel-Latif), mbahgatriad @yahoo.com, [email protected] (M. Bahgat). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.022
aria immitis infected dogs after DEC treatment were eosinophils (Sutton et al., 1985). Difference in the microfilaricidal effect of DEC was referred by others to be due to different phagocytic activity of liver kupffer cells (Hayashi et al., 1983). The microfilaricidal effect of the drug was accompanied by an increased occurrence of Litomosoides carinii larvae in the lymph nodes and peritoneal cavity (Zahner and Weidner, 1983). Following DEC treatment, the induced cellular immune response to mf was characterized by an increased activity of lymphocytes (Piessens et al., 1981; Mistry and Subrahmanyam, 1986; Sartono et al., 1995), of eosinophils (Ottesen and Weller, 1979; Gopinath et al., 2000), macrophages (Tyagi et al., 1986) and natural killer cells (Pedersen et al., 1987). Recently, the success of mass DEC administration over 8 years was reported by Hooper et al. (2009) that prevented the spread of filarial infection to 6.6 million newborns, stopped the progression to clinical morbidity in 9.5 million individuals already infected and drastically reduced the burden of several co-infections. The prevalence of the animal filarial parasite Setaria equina among Egyptian equines and the extents of cross reaction between antigens prepared from such parasite with sera from Wuchereria bancrofti infected Egyptian patients were recently reported (Bahgat et al., in press). Herein, albino rats were used as experimental hosts for S. equina infection to investigate the effect of the DEC treatment on the parasite mf.
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2. Materials and methods 2.1. Parasite collection While equines were dissected, adult female S. equina worms were collected in peritoneal fluid and transferred to the laboratory. Worms were washed three times with warm phosphate buffered saline containing antibiotics (1 PBS; 100 U/ml penicillin, 100 lg/ml streptomycin). 2.2. Experimental infection All the experimental animal work was carried out according to the guide lines and the standard regulations of the Faculty of Science, Beni-Suef University and the National Research Center of Egypt. Male white albino rats (250–300 g each) were pre-injected with atropine sulfate (0.4 mg/kg) to manage possible parasympathetic side effects of anesthesia. Animals were anaesthetized by intramuscular injection with sodium thiopental (100 mg/kg; Biochemie GmbH, Vienna-Austria). Three gravid female worms were implanted in the peritoneal cavity of each rat through a small slit that was immediately stitched with surgical catgut under aseptic condition (Ghosh et al., 1993; Mukhopadhyay et al., 1996) followed by once daily topical administration of a skin ointment (Terramycin; Pfizer, New York, USA) on the stitched region till complete recovery of the wound. After 38 days, blood was collected from orbital plexus on sodium citrate and conventional thick smears were examined for presence of mf using light microscope. 2.3. In vivo effect of DEC on S. equina mf One week after implanting S. equina gravid female worms in the peritoneal cavities of 30 male albino rats, blood samples were collected from the orbit plexus of individual animals and thick smears (20 ll) were examined. Microfilariae were counted in triplicates and the mean count was calculated. Eight rats were treated by intravenous (i.v.) injection with DEC dissolved in normal saline at a dosage of 25 mg/kg in 0.5 ml/rat (Schardein et al., 1968) while eight control animals received only 0.5 ml normal saline/rat. After treated and control animals had been sacrificed at 45, 300 and 480 min, blood was collected from individual animals and tested for mf count. Lungs, livers, spleens and kidneys were excised from treated and control animals and examined for trapped mf and any histologic changes using light microscope. 2.4. Statistical analysis The differences in the means of the mf counts between the control and DEC-treated rat groups were calculated by the Student’s t test using the practistat statistics program (Ashcroft-Pereira, London, UK). 3. Results 3.1. Changes in blood mf counts in response to treatment with DEC One to two weeks after rat infection with S. equina worms, blood mf count reached 3.5 103/ml. DEC treatment caused a more significant decline (95%, P < 0.001) in the mf counts at 45 min, less significant at 300 min (69%, P < 0.05) and non-significant at 480 min (67%, P > 0.05) (Fig. 1). 3.2. Changes in trapped mf and animal tissues post-treatment At 45 min, 5 and 8 h after treating microfilaremic rats with either DEC or normal saline, histologic sections from livers, lungs,
Fig. 1. Pattern changes in percentage microfilaremia of Setaria equina (mean averages) over time points (45 min, 300 and 480 min) in treated and control groups. DEC treatment caused a highly significant decline (95%, P < 0.001) in the mf counts at 45 min, less significant at 300 min (69%, P < 0.05) and non-significant at 480 min (67%, P > 0.05).
kidneys and spleens were excised and examined by light microscopy.
3.2.1. Liver At 45 min, 5 and 8 h post administration of saline, mf were scarce and found only in blood vessels (Fig. 2A, C and E). At 45 min post administration of DEC, higher counts of abnormally appearing mf were mainly recorded in the hepatic sinusoids adjacent to aggregates of kupffer cells (Fig. 2B) with lysis in their cellular cytoplasm. However, many inflammatory foci (IF) appeared in liver sections at 5 h post DEC administration (Fig 2D). Inside each focus, one or two degenerated mf were visualized surrounded by different types of leukocytes, predominantly eosinophils. At 8 h post DEC treatment, degenerated mf were observed inside one of the hepatic sinusoids without any surrounding leukocytes (Fig. 2F).
3.2.2. Lung At 45 min, 5 and 8 h post saline administration, the mf were scarce and intact with enveloping sheath (Fig. 3A, C and E). At 45 min post DEC treatment, higher counts of mf that lost their sheath were observed (Fig. 3B). At both 5 and 8 h post DEC administration, mf were seen degenerated and surrounded with leukocytes (Fig. 3D and F).
3.2.3. Kidney In both sections of control and treated rats, mf were only found in the cortical tissue including the glomerular capillaries causing severe damage for some glomeruli. At all time points after saline administration, the glomerular capillaries appeared occluded with an increase in mesangial cells and matrix, while the mf were intact (Fig. 4A, C and E). In DEC-treated rats, higher numbers of mf as accompanied by decrease in matrix were observed than in saline given animals (Fig. 4B, D and F). Only at 5 h post DEC treatment, mf in the glomerular capillaries appeared degenerated (Fig. 4D).
3.2.4. Spleen At 45 min and 5 h post saline or DEC administration, mf appeared less damaged inside a clear space-like vacuoles with no surrounding cells (Fig. 5A–D). However, mf were observed to be degenerated and surrounded with cells inside the clear space only at 8 h post DEC treatment (Fig. 5E and F).
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Fig. 2. Changes in trapped mf and liver tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; BV, blood vessel; ERY, erythrocyte; KC, kupffer cell; LC, lysed cytoplasm; lek, leukocytes; IF, inflammatory focus; eos, eosinophils; HS, hepatic sinusoid; small arrow denotes to a breaking damage in microfilaria. The used magnification was 1000.
4. Discussion The obvious drop in mf counts recorded in the present work at 45 min, 5 and 8 h post DEC administration in comparison to saline given animals agrees with results obtained by Schardein et al. (1968) who monitored drop in mf counts and ultrastructural changes at times close to 45 min and 5 h post DEC treatment of gerbils infected with L. carinii in comparison to control animals. Our attempt to monitor the DEC effects, at later time point, 8 h was to investigate if this will add any new information to the previously published data. The early migration of the larvae from the circulation following DEC treatment was explained by Zahner and Weidner (1983) to be a result of mf activation by the drug and their new locations depend on the site where they were living. Alternatively, Maizels and Denham (1992) suggested that DEC blocks the mf-secreted prostaglandin E2 and prostacyclins 12, thus, leading to a sufficient constriction of the capillaries to impede the passage of mf leading to their subsequent disappearance from the circulation.
The pronounced decrease in blood mf counts at 45 min after DEC treatment and their increase at later time points we recorded is consistent with that of Zahner and Weidner (1983) who worked on L. carinii. The same finding was also reported by Horii and Aoki (1997) upon studying DEC effect on i.v. implanted mf of Brugia pahangi in rats, nonetheless, there is still a debate for the reason for such phenomenon (Weiner and Soulsby, 1982; Zahner and Weidner, 1983; Kani et al., 1983; Dixit et al., 2009). It is widely accepted that DEC initially induces accumulation of mf in various organs, especially the liver (Hawking et al., 1950; Mitsui et al., 1966). In our results, the marked decrease of blood mf at 45 min post-treatment was associated with visualizing abnormal mf with lysed cellular cytoplasm in the liver sinusoidal spaces without points of cellular attachment but with adjacent kupffer cells. This agrees with the observations of Schardein et al. (1968) and Mehlhorn et al. (1981) upon examining the in vivo effect of DEC on both Dipetalonema viteae and L. carinii mf and reflects the major role might be played by the kupffer cells in mf lysis after DEC treatment. In our control rats, mf were only found
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Fig. 3. Changes in trapped mf and lung tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; SH, sheath; lek, leukocyte; ERY, erythrocytes; BV, blood vessel. The used magnification was 1000.
in the tissue blood capillaries. This agrees with the report of Shigeno et al. (2006) who found more mf in blood vessels than in capillary/tissue areas 30 min after saline administration to infected jirds with B. pahangi. Recording degenerated mf inside a high number of hepatic IF at 300 min post-treatment which possessed various types of leukocytes with prominent existence of eosinophils in our hands, agrees with previous studies where different filarial species and animal models were used (Schardein et al., 1968; Piessens and Beldekas, 1979; Fanning and Kazura, 1985; Sutton et al., 1985; Gopinath et al., 2000). Moreover, previous studies on the kinetics of macrophage adherence to mf of both Dipetalonema
vitae and Breinlia macropi and subsequent cytotoxicity were found to be through encircling mf by the protoplasmic walls where secretory granules were seen adjacent to parasite surface (Ouaissi et al., 1981; Yen et al., 1986). The efficacy of DEC to induce cellular adherence to mf could be explained to be due to its direct effect on such parasite stage that causes surface changes and release of mf antigens thus enhancing antibody or complement mediated cellular adherence (Schardein et al., 1968; Piessens and Beldekas, 1979; Carme et al., 1981; Zahner, 1983; Silva et al., 2006). In the present work, a marked degeneration of mf in the hepatic sinusoids was noticed at 480 min post DEC
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Fig. 4. Changes in trapped mf and kidney tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; GL, glomerulus; BS, Bowman’s space; lek, leukocytes; M, matrix. The used magnification was 1000.
treatment with no evidenced adherent cells. In consistence, Zahner et al. (1978) observed a decrease in the number of cell adherent mf at the same time point. Exsheathment of mf was observed in the lung at 45 min posttreatment without visualizing any attached cells agrees with previous report on the low DEC-dependent cell attachment in the lungs compared to other organs (Zahner et al., 1978). DEC was found to ameliorate inflammatory reactions in the lung through different mechanisms (Orange et al., 1971; Magnussen et al., 1995; Florencio et al., 2005; Queto et al., 2009). Although the loss of mf sheath after 45 min of DEC treatment was independent of cellular adherence in both liver and lung tissues, cellular adherence to degenerated mf was evident at 300 min post-treatment that might allow the assumption that DEC exerts a reversal effect
by activation of cellular response at the later time points posttreatment. Thus, the inhibitory effect of DEC for the release of inflammatory mediators (Kanesa-thasan et al., 1991) seems to be no more existing at, 300 min, later time points post-treatment. The appearance of degenerated mf with surrounding leukocytes in the lung tissues at late time point post DEC administration highlights the lung as an essential organ where immune attack on mf occurs (Zahner and Weidner, 1983). At 480 min post-treatment, appearance of degenerated mf and adherent cells in our results is consistent with the previous observation of Zahner et al. (1978) who observed an increase in cell adhesion from the 4th to the 8th hour after treatment. The observed damage for the glomerular capillaries in both test and control rats at all time points is not surprising in the light of
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Fig. 5. Changes in trapped mf and spleen tissue post-treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post normal saline administration, respectively. B, D and F were sections from post DEC administration at 45 min, 5 and 8 h, respectively. MF, microfilaria; CS, clear space; lek, leukocytes. The used magnification was 1000.
previous report by Dreyer et al. (1992) where some of untreated asymptomatic microfilaremic patients had hematuria and proteinuria reflecting severe renal pathology. The most likely explanation is the mechanical damage of mf to glomerular capillaries and the presence of immune complexes in the renal glomeruli (Abramowsky et al., 1981; Dixit et al., 2007). The increase in the mf counts and decrease in the glomeruli matrix observed at all time points following DEC treatment by ourselves in comparison to control
animals agree with previous reports on Onchocerca volvulus and W. bancrofti (Duke et al., 1975; Dreyer et al., 1992). The decrease in matrix expansion may reflect decrease in the deposition of immune complexes in kidney glomeruli due to DEC treatment as the increase in glomerular mesangium was previously referred to the deposition of immune complexes in the glomerular capillaries (Klei et al., 1974; Casey and Splitter, 1975; McCarthy et al., 1995). Our observation for degenerated mf at 300 min post-treatment
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with DEC means that the kidney shares with the liver and lung the ability to eliminate mf from circulation that supports the previous finding of Zahner et al. (1978). Although previous results have outlined the significance of inflammatory cell infiltrates in the glomerulus after treatment with DEC (Meyers et al., 1977; McManus and Pulliam, 1984), no leukocytes were observed to be adherent to mf in our hands. In fact, cellular adhesion was reported by Zahner and coworkers (1978) to occur in the kidney tissues at earlier time points post therapy and its peak was recorded at 16 h post DEC administration. In the spleen, degenerated mf surrounded by cells were only observed in the present work at 480 min after treatment, indicating the involvement of the spleen in mf elimination only at late time points post-treatment contradicting previous findings of Zahner et al. (1978). This suggests an effective involvement of the spleen in the elimination of L. carinii mf by an earlier presence of adherent cells at 1 h post DEC administration. Our interpretation for such discrepancy could rely on studying different filarial species, using different animal model and applying the drug through different route of administration. Interestingly, DEC was previously found to inhibit in vitro adherence of rat spleen cells to L. carinii mf (Mehta et al., 1980). In conclusion, a single i.v. administration of DEC to infected albino rats caused an earlier sharp decrease in mf count followed by an increase. Efficacy of the drug was associated with an accumulation of mf in body organs like liver, lung, kidney and spleen, yet, the cellular adherence to mf was only observed at later time points. Results of the present report highlight the essential role of DEC to trap mf in host tissues making them more vulnerable to the attack by immune cells. Future perspectives are to investigate possible involvements of both antibodies and complement in cellular adherence and parasite killing. Acknowledgments We are grateful to the Pathology Unit at the National Cancer Institute of Egypt for providing technical help for preparing and mounting tissue sections for histological examination. Special thanks are due to Prof. Dr. Mohamed Ahmed Ali, the Head of the Environmental Virology Laboratory at the National Research Center of Egypt for providing the light microscopy and photography facilities. References Abramowsky, C.R., Powers, K.G., Aikawa, M., Swinehart, G., 1981. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. American Journal of Pathology 104, 1–12. Bahgat, M., Saad, A.H., El-Shahawi, G.A., Gad, A.M., Ramzy, R.M., Ruppel, A., AbdelLatif, M., in press. Cross-reaction of antigen preparations from adult and larval stages of the parasite Setaria equina with sera from infected humans with Wuchereria bancrofti. Eastern Mediterranean Health Journal. Carme, B., Richard-Lenoble, D., Smith, M., Pontal, P., Gentilini, M., 1981. Litomosoides carinii infection in cotton rats: evolution of microfilaremia before and after treatment with diethylcarbamazine and suramin. Transaction of Royal Society of Tropical Medicine and Hygiene 75, 418–420. Casey, H.W., Splitter, G.A., 1975. Membranous glomerulonephritis in dogs infected with Dirofilaria immitis. Veterinary Pathology 12, 111–117. Dixit, V., Subhadra, A.V., Bisen, P.S., Harinath, B.C., Prasad, G.B., 2007. Antigenspecific immune complexes in urine of patients with lymphatic filariasis. Journal of Clinical Laboratory Analysis 21, 46–48. Dixit, V., Gupta, A.K., Prasad, G.B., 2009. Interruption of annual single dose DEC regimen administration: impact on Wuchereria bancrofti microfilaraemia, vector infection and infectivity rates. The Journal of Communicable Diseases 41, 25– 31. Dreyer, G., Ottesen, E.A., Galdino, E., Andrade, L., Rocha, A., Medeiros, Z., Moura, I., Casimiro, I., Beliz, F., Coutinho, A., 1992. Renal abnormalities in microfilaremic patients with Bancroftian filariasis. American Journal of Tropical Medicine and Hygiene 46, 745–751. Duke, B.O., Moore, P.J., Vincelette, J., 1975. Factors influencing the passage of Onchocerca volvulus microfilariae into the urine. Tropenmedizin und Parasitologie 26, 449–468.
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adult forms of Litomosoides chagasfilhoi and in the free-living nematode Caenorhabditis elegans. Micron 37, 666–674. Sutton, R.H., Atwell, R.B., Boreham, P.F.L., 1985. Liver changes, following diethylcarbamazine administration, in microfilaremic dogs infected with Dirofilaria immitis. Veterinary Pathology 22, 177–183. Tyagi, K., Murthy, P.K., Sen, A.B., 1986. Effect of some known antifilarials on the immune responses of Mastomys natalensis infected with Brugia malayi. Indian Journal of Medical Research 83, 155–161. Weiner, D.J., Soulsby, E.J., 1982. Litomosoides carinii: effect of diethylcarbamazine on microfilaremias of Mastomys natalensis harboring old infections, new infections, and transfused microfilariae. Journal of Parasitology 68, 1105–1109. Weiner, D.J., Abraham, D., D’Antonio, R.D., 1986. The effect of diethylcarbamazine on microfilariae of Litomosoides carinii in vitro and in vivo. Zeitschrift für Parasitenkund 72, 805–813. Yen, P.K.F., Holt, P.G., Stanley, N.F., Papadi-Mitriou, J.M., Robertson, T., 1986. In vitro antibody-mediated macrophage activity on Breinlia macropi microfilariae. II. Ultrastructural and video recording investigations of adherence and cytotoxicity. Parasite Immunology 8, 201–216. Zahner, H., 1983. Diethylcarbamazine dependent, complement mediated adherence and cytotoxicity of cells on microfilariae of Litomosoides carinii. Acta Tropica 40, 129–138. Zahner, H., Weidner, E., 1983. Occurrence of microfilariae in various hollow organs and in the peritoneal cavity after treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine and haloxon. Zeitschrift für Parasitenkund 69, 387–392. Zahner, H., Soulsby, E.J.L., Weidner, E., Lammler, G., 1978. Effect of diethylcarbamazine on the microfilariae of Litomosoides carinii in Mastomys natalensis: dynamics of cell adhesion, immobilization and elimination of microfilariae. Tropenmedizin und Parasitologie 29, 15–26.
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Setaria equina: In vivo effect of diethylcarbamazine citrate on microfilariae in albino rats G.A. El-Shahawi a, M. Abdel-Latif a,*, A.H. Saad b, M. Bahgat c,** a
Department of Zoology, Faculty of Science, Beni-Suef University, Egypt Department of Zoology, Faculty of Science, Cairo University, Egypt c Therapeutic Chemistry Department, Infectious Diseases and Immunology Laboratory, The Center of Excellence for Advanced Sciences, The National Research Center, Dokki, Cairo 12311, Egypt b
a r t i c l e
i n f o
Article history: Received 29 October 2009 Received in revised form 17 May 2010 Accepted 15 June 2010 Available online 18 June 2010 Keywords: Setaria equina Albino rats Experimental infection Diethylcarbamazine citrate Organs
a b s t r a c t Although diethylcarbamazine citrate (DEC) is successful drug in eliminating human filariasis, yet, its mode of action is still debatable. Herein, the effect of DEC to treat albino rats infected with the animal filarial parasite Setaria equina was tested. Microfilarial (mf) counts and sections from liver, lung, kidney as well as spleen were investigated at different time points after treatment by light microscopy. After 45 and 300 min of treatment, a significant decrease in blood mf was observed accompanied by adherence of degenerated mf to both kupffer cells and leukocyte in liver sections. In lung sections, loss of sheath was observed at 45 min, while degeneration was observed at later time points. In kidney sections, more mf counts and less matrix were observed in the glomeruli at all time points after treatment. Degenerated mf were observed in spleen sections only at, late time point, 480 min after treatment. In conclusion, one of the possible mechanisms by which DEC reduces blood microfilarial count is trapping larvae in organs and killing them through cellular adherence. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction DEC is a piperazine derivative having both microfilaricidal and macrofilaricidal activities. The success of control programs depends on whether the adult worms are killed or temporarily sterilized and the extent of microfilarial clearance (Eberhard et al., 1997). Therapeutic efficacy of DEC was described more than 50 years ago, yet, its mode of action is still debatable (Hewitt et al., 1947; Santiago-Stevenson et al., 1947). Treating infected experimental animals with filarial parasites by DEC caused elimination of mf from circulation (Zahner et al., 1978; Weiner et al., 1986; Mukhopadhyay et al., 1996) that was referred to the highest rate of cellular adherence, phagocytosis and subsequent lysis of mf in the sinusoids and parenchymal inflammatory foci of the liver (Schardein et al., 1968; Zahner et al., 1978; Piessens and Beldekas, 1979; Fanning and Kazura, 1985). The predominant inflammatory cells in the liver of Dirofil-
* Corresponding author. Fax: +20 822334551. ** Correspondence to: M. Bahgat, Current address: Department of Infection Genetics, the Helmholtz Center for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. E-mail addresses: [email protected] (M. Abdel-Latif), mbahgatriad @yahoo.com, [email protected] (M. Bahgat). 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.022
aria immitis infected dogs after DEC treatment were eosinophils (Sutton et al., 1985). Difference in the microfilaricidal effect of DEC was referred by others to be due to different phagocytic activity of liver kupffer cells (Hayashi et al., 1983). The microfilaricidal effect of the drug was accompanied by an increased occurrence of Litomosoides carinii larvae in the lymph nodes and peritoneal cavity (Zahner and Weidner, 1983). Following DEC treatment, the induced cellular immune response to mf was characterized by an increased activity of lymphocytes (Piessens et al., 1981; Mistry and Subrahmanyam, 1986; Sartono et al., 1995), of eosinophils (Ottesen and Weller, 1979; Gopinath et al., 2000), macrophages (Tyagi et al., 1986) and natural killer cells (Pedersen et al., 1987). Recently, the success of mass DEC administration over 8 years was reported by Hooper et al. (2009) that prevented the spread of filarial infection to 6.6 million newborns, stopped the progression to clinical morbidity in 9.5 million individuals already infected and drastically reduced the burden of several co-infections. The prevalence of the animal filarial parasite Setaria equina among Egyptian equines and the extents of cross reaction between antigens prepared from such parasite with sera from Wuchereria bancrofti infected Egyptian patients were recently reported (Bahgat et al., in press). Herein, albino rats were used as experimental hosts for S. equina infection to investigate the effect of the DEC treatment on the parasite mf.
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2. Materials and methods 2.1. Parasite collection While equines were dissected, adult female S. equina worms were collected in peritoneal fluid and transferred to the laboratory. Worms were washed three times with warm phosphate buffered saline containing antibiotics (1 PBS; 100 U/ml penicillin, 100 lg/ml streptomycin). 2.2. Experimental infection All the experimental animal work was carried out according to the guide lines and the standard regulations of the Faculty of Science, Beni-Suef University and the National Research Center of Egypt. Male white albino rats (250–300 g each) were pre-injected with atropine sulfate (0.4 mg/kg) to manage possible parasympathetic side effects of anesthesia. Animals were anaesthetized by intramuscular injection with sodium thiopental (100 mg/kg; Biochemie GmbH, Vienna-Austria). Three gravid female worms were implanted in the peritoneal cavity of each rat through a small slit that was immediately stitched with surgical catgut under aseptic condition (Ghosh et al., 1993; Mukhopadhyay et al., 1996) followed by once daily topical administration of a skin ointment (Terramycin; Pfizer, New York, USA) on the stitched region till complete recovery of the wound. After 38 days, blood was collected from orbital plexus on sodium citrate and conventional thick smears were examined for presence of mf using light microscope. 2.3. In vivo effect of DEC on S. equina mf One week after implanting S. equina gravid female worms in the peritoneal cavities of 30 male albino rats, blood samples were collected from the orbit plexus of individual animals and thick smears (20 ll) were examined. Microfilariae were counted in triplicates and the mean count was calculated. Eight rats were treated by intravenous (i.v.) injection with DEC dissolved in normal saline at a dosage of 25 mg/kg in 0.5 ml/rat (Schardein et al., 1968) while eight control animals received only 0.5 ml normal saline/rat. After treated and control animals had been sacrificed at 45, 300 and 480 min, blood was collected from individual animals and tested for mf count. Lungs, livers, spleens and kidneys were excised from treated and control animals and examined for trapped mf and any histologic changes using light microscope. 2.4. Statistical analysis The differences in the means of the mf counts between the control and DEC-treated rat groups were calculated by the Student’s t test using the practistat statistics program (Ashcroft-Pereira, London, UK). 3. Results 3.1. Changes in blood mf counts in response to treatment with DEC One to two weeks after rat infection with S. equina worms, blood mf count reached 3.5 103/ml. DEC treatment caused a more significant decline (95%, P < 0.001) in the mf counts at 45 min, less significant at 300 min (69%, P < 0.05) and non-significant at 480 min (67%, P > 0.05) (Fig. 1). 3.2. Changes in trapped mf and animal tissues post-treatment At 45 min, 5 and 8 h after treating microfilaremic rats with either DEC or normal saline, histologic sections from livers, lungs,
Fig. 1. Pattern changes in percentage microfilaremia of Setaria equina (mean averages) over time points (45 min, 300 and 480 min) in treated and control groups. DEC treatment caused a highly significant decline (95%, P < 0.001) in the mf counts at 45 min, less significant at 300 min (69%, P < 0.05) and non-significant at 480 min (67%, P > 0.05).
kidneys and spleens were excised and examined by light microscopy.
3.2.1. Liver At 45 min, 5 and 8 h post administration of saline, mf were scarce and found only in blood vessels (Fig. 2A, C and E). At 45 min post administration of DEC, higher counts of abnormally appearing mf were mainly recorded in the hepatic sinusoids adjacent to aggregates of kupffer cells (Fig. 2B) with lysis in their cellular cytoplasm. However, many inflammatory foci (IF) appeared in liver sections at 5 h post DEC administration (Fig 2D). Inside each focus, one or two degenerated mf were visualized surrounded by different types of leukocytes, predominantly eosinophils. At 8 h post DEC treatment, degenerated mf were observed inside one of the hepatic sinusoids without any surrounding leukocytes (Fig. 2F).
3.2.2. Lung At 45 min, 5 and 8 h post saline administration, the mf were scarce and intact with enveloping sheath (Fig. 3A, C and E). At 45 min post DEC treatment, higher counts of mf that lost their sheath were observed (Fig. 3B). At both 5 and 8 h post DEC administration, mf were seen degenerated and surrounded with leukocytes (Fig. 3D and F).
3.2.3. Kidney In both sections of control and treated rats, mf were only found in the cortical tissue including the glomerular capillaries causing severe damage for some glomeruli. At all time points after saline administration, the glomerular capillaries appeared occluded with an increase in mesangial cells and matrix, while the mf were intact (Fig. 4A, C and E). In DEC-treated rats, higher numbers of mf as accompanied by decrease in matrix were observed than in saline given animals (Fig. 4B, D and F). Only at 5 h post DEC treatment, mf in the glomerular capillaries appeared degenerated (Fig. 4D).
3.2.4. Spleen At 45 min and 5 h post saline or DEC administration, mf appeared less damaged inside a clear space-like vacuoles with no surrounding cells (Fig. 5A–D). However, mf were observed to be degenerated and surrounded with cells inside the clear space only at 8 h post DEC treatment (Fig. 5E and F).
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Fig. 2. Changes in trapped mf and liver tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; BV, blood vessel; ERY, erythrocyte; KC, kupffer cell; LC, lysed cytoplasm; lek, leukocytes; IF, inflammatory focus; eos, eosinophils; HS, hepatic sinusoid; small arrow denotes to a breaking damage in microfilaria. The used magnification was 1000.
4. Discussion The obvious drop in mf counts recorded in the present work at 45 min, 5 and 8 h post DEC administration in comparison to saline given animals agrees with results obtained by Schardein et al. (1968) who monitored drop in mf counts and ultrastructural changes at times close to 45 min and 5 h post DEC treatment of gerbils infected with L. carinii in comparison to control animals. Our attempt to monitor the DEC effects, at later time point, 8 h was to investigate if this will add any new information to the previously published data. The early migration of the larvae from the circulation following DEC treatment was explained by Zahner and Weidner (1983) to be a result of mf activation by the drug and their new locations depend on the site where they were living. Alternatively, Maizels and Denham (1992) suggested that DEC blocks the mf-secreted prostaglandin E2 and prostacyclins 12, thus, leading to a sufficient constriction of the capillaries to impede the passage of mf leading to their subsequent disappearance from the circulation.
The pronounced decrease in blood mf counts at 45 min after DEC treatment and their increase at later time points we recorded is consistent with that of Zahner and Weidner (1983) who worked on L. carinii. The same finding was also reported by Horii and Aoki (1997) upon studying DEC effect on i.v. implanted mf of Brugia pahangi in rats, nonetheless, there is still a debate for the reason for such phenomenon (Weiner and Soulsby, 1982; Zahner and Weidner, 1983; Kani et al., 1983; Dixit et al., 2009). It is widely accepted that DEC initially induces accumulation of mf in various organs, especially the liver (Hawking et al., 1950; Mitsui et al., 1966). In our results, the marked decrease of blood mf at 45 min post-treatment was associated with visualizing abnormal mf with lysed cellular cytoplasm in the liver sinusoidal spaces without points of cellular attachment but with adjacent kupffer cells. This agrees with the observations of Schardein et al. (1968) and Mehlhorn et al. (1981) upon examining the in vivo effect of DEC on both Dipetalonema viteae and L. carinii mf and reflects the major role might be played by the kupffer cells in mf lysis after DEC treatment. In our control rats, mf were only found
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Fig. 3. Changes in trapped mf and lung tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; SH, sheath; lek, leukocyte; ERY, erythrocytes; BV, blood vessel. The used magnification was 1000.
in the tissue blood capillaries. This agrees with the report of Shigeno et al. (2006) who found more mf in blood vessels than in capillary/tissue areas 30 min after saline administration to infected jirds with B. pahangi. Recording degenerated mf inside a high number of hepatic IF at 300 min post-treatment which possessed various types of leukocytes with prominent existence of eosinophils in our hands, agrees with previous studies where different filarial species and animal models were used (Schardein et al., 1968; Piessens and Beldekas, 1979; Fanning and Kazura, 1985; Sutton et al., 1985; Gopinath et al., 2000). Moreover, previous studies on the kinetics of macrophage adherence to mf of both Dipetalonema
vitae and Breinlia macropi and subsequent cytotoxicity were found to be through encircling mf by the protoplasmic walls where secretory granules were seen adjacent to parasite surface (Ouaissi et al., 1981; Yen et al., 1986). The efficacy of DEC to induce cellular adherence to mf could be explained to be due to its direct effect on such parasite stage that causes surface changes and release of mf antigens thus enhancing antibody or complement mediated cellular adherence (Schardein et al., 1968; Piessens and Beldekas, 1979; Carme et al., 1981; Zahner, 1983; Silva et al., 2006). In the present work, a marked degeneration of mf in the hepatic sinusoids was noticed at 480 min post DEC
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Fig. 4. Changes in trapped mf and kidney tissue after treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post administration of saline, respectively. B, D and F were sections from post administration of DEC at 45 min, 5 and 8 h, respectively. MF, microfilaria; GL, glomerulus; BS, Bowman’s space; lek, leukocytes; M, matrix. The used magnification was 1000.
treatment with no evidenced adherent cells. In consistence, Zahner et al. (1978) observed a decrease in the number of cell adherent mf at the same time point. Exsheathment of mf was observed in the lung at 45 min posttreatment without visualizing any attached cells agrees with previous report on the low DEC-dependent cell attachment in the lungs compared to other organs (Zahner et al., 1978). DEC was found to ameliorate inflammatory reactions in the lung through different mechanisms (Orange et al., 1971; Magnussen et al., 1995; Florencio et al., 2005; Queto et al., 2009). Although the loss of mf sheath after 45 min of DEC treatment was independent of cellular adherence in both liver and lung tissues, cellular adherence to degenerated mf was evident at 300 min post-treatment that might allow the assumption that DEC exerts a reversal effect
by activation of cellular response at the later time points posttreatment. Thus, the inhibitory effect of DEC for the release of inflammatory mediators (Kanesa-thasan et al., 1991) seems to be no more existing at, 300 min, later time points post-treatment. The appearance of degenerated mf with surrounding leukocytes in the lung tissues at late time point post DEC administration highlights the lung as an essential organ where immune attack on mf occurs (Zahner and Weidner, 1983). At 480 min post-treatment, appearance of degenerated mf and adherent cells in our results is consistent with the previous observation of Zahner et al. (1978) who observed an increase in cell adhesion from the 4th to the 8th hour after treatment. The observed damage for the glomerular capillaries in both test and control rats at all time points is not surprising in the light of
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Fig. 5. Changes in trapped mf and spleen tissue post-treatment of microfilaremic rats with either DEC or normal saline. A, C and E represent sections from 45 min, 5 and 8 h post normal saline administration, respectively. B, D and F were sections from post DEC administration at 45 min, 5 and 8 h, respectively. MF, microfilaria; CS, clear space; lek, leukocytes. The used magnification was 1000.
previous report by Dreyer et al. (1992) where some of untreated asymptomatic microfilaremic patients had hematuria and proteinuria reflecting severe renal pathology. The most likely explanation is the mechanical damage of mf to glomerular capillaries and the presence of immune complexes in the renal glomeruli (Abramowsky et al., 1981; Dixit et al., 2007). The increase in the mf counts and decrease in the glomeruli matrix observed at all time points following DEC treatment by ourselves in comparison to control
animals agree with previous reports on Onchocerca volvulus and W. bancrofti (Duke et al., 1975; Dreyer et al., 1992). The decrease in matrix expansion may reflect decrease in the deposition of immune complexes in kidney glomeruli due to DEC treatment as the increase in glomerular mesangium was previously referred to the deposition of immune complexes in the glomerular capillaries (Klei et al., 1974; Casey and Splitter, 1975; McCarthy et al., 1995). Our observation for degenerated mf at 300 min post-treatment
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with DEC means that the kidney shares with the liver and lung the ability to eliminate mf from circulation that supports the previous finding of Zahner et al. (1978). Although previous results have outlined the significance of inflammatory cell infiltrates in the glomerulus after treatment with DEC (Meyers et al., 1977; McManus and Pulliam, 1984), no leukocytes were observed to be adherent to mf in our hands. In fact, cellular adhesion was reported by Zahner and coworkers (1978) to occur in the kidney tissues at earlier time points post therapy and its peak was recorded at 16 h post DEC administration. In the spleen, degenerated mf surrounded by cells were only observed in the present work at 480 min after treatment, indicating the involvement of the spleen in mf elimination only at late time points post-treatment contradicting previous findings of Zahner et al. (1978). This suggests an effective involvement of the spleen in the elimination of L. carinii mf by an earlier presence of adherent cells at 1 h post DEC administration. Our interpretation for such discrepancy could rely on studying different filarial species, using different animal model and applying the drug through different route of administration. Interestingly, DEC was previously found to inhibit in vitro adherence of rat spleen cells to L. carinii mf (Mehta et al., 1980). In conclusion, a single i.v. administration of DEC to infected albino rats caused an earlier sharp decrease in mf count followed by an increase. Efficacy of the drug was associated with an accumulation of mf in body organs like liver, lung, kidney and spleen, yet, the cellular adherence to mf was only observed at later time points. Results of the present report highlight the essential role of DEC to trap mf in host tissues making them more vulnerable to the attack by immune cells. Future perspectives are to investigate possible involvements of both antibodies and complement in cellular adherence and parasite killing. Acknowledgments We are grateful to the Pathology Unit at the National Cancer Institute of Egypt for providing technical help for preparing and mounting tissue sections for histological examination. Special thanks are due to Prof. Dr. Mohamed Ahmed Ali, the Head of the Environmental Virology Laboratory at the National Research Center of Egypt for providing the light microscopy and photography facilities. References Abramowsky, C.R., Powers, K.G., Aikawa, M., Swinehart, G., 1981. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. American Journal of Pathology 104, 1–12. Bahgat, M., Saad, A.H., El-Shahawi, G.A., Gad, A.M., Ramzy, R.M., Ruppel, A., AbdelLatif, M., in press. Cross-reaction of antigen preparations from adult and larval stages of the parasite Setaria equina with sera from infected humans with Wuchereria bancrofti. Eastern Mediterranean Health Journal. Carme, B., Richard-Lenoble, D., Smith, M., Pontal, P., Gentilini, M., 1981. Litomosoides carinii infection in cotton rats: evolution of microfilaremia before and after treatment with diethylcarbamazine and suramin. Transaction of Royal Society of Tropical Medicine and Hygiene 75, 418–420. Casey, H.W., Splitter, G.A., 1975. Membranous glomerulonephritis in dogs infected with Dirofilaria immitis. Veterinary Pathology 12, 111–117. Dixit, V., Subhadra, A.V., Bisen, P.S., Harinath, B.C., Prasad, G.B., 2007. Antigenspecific immune complexes in urine of patients with lymphatic filariasis. Journal of Clinical Laboratory Analysis 21, 46–48. Dixit, V., Gupta, A.K., Prasad, G.B., 2009. Interruption of annual single dose DEC regimen administration: impact on Wuchereria bancrofti microfilaraemia, vector infection and infectivity rates. The Journal of Communicable Diseases 41, 25– 31. Dreyer, G., Ottesen, E.A., Galdino, E., Andrade, L., Rocha, A., Medeiros, Z., Moura, I., Casimiro, I., Beliz, F., Coutinho, A., 1992. Renal abnormalities in microfilaremic patients with Bancroftian filariasis. American Journal of Tropical Medicine and Hygiene 46, 745–751. Duke, B.O., Moore, P.J., Vincelette, J., 1975. Factors influencing the passage of Onchocerca volvulus microfilariae into the urine. Tropenmedizin und Parasitologie 26, 449–468.
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