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In vitro and In vivo Anti-Filarial Effect of Tetracycline/Doxycycline.

Lymphatic filariasis (LF) is a disease of considerable socio-economic burden in the tropics and sub-tropics and is caused by Wuchereria bancrofti, Brugia malayi and B. temori. Wolbachia endosymbiontic bacteria are found in mutualistic relationship in many filarial nematodes infecting animals and humans including W. bancrofti. Currently, the estimated 68 million people infected, among them, 36 million are microfilaria carriers and 40 million are symptomatic (1). Additionally, 946 million people live in areas of southeast Asia and sub-Saharan Africa are at risk of infection (1). Anti-filarial chemotherapy is associated with systemic adverse reaction, due to release of microfilariae and Wolbachia bacteria into the blood. The available standard chemotherapy kills only the microfilariae and their macrofilaricidal function is not established either in vitro or in vivo. In recent past anti-Wolbachia antibiotics treatment has become a novel approach to treat lymphatic filariasis; these antibiotics inhibit worm development, embryogenesis, fertility and viability. The studies have demonstrated sub-lethal effect of antibiotics on filarial worms and this is due to effect on the Wolbachia (2,3,4). Anti-Wolbachia treatment studies of tetracycline on animal models revealed the reduction in worm burden and blocking molting of infective stage larvae (L3 to L4 and L4 to L5) as well as causes distortion of male/ female sex ratio (5,6,7).

Studies carried out in human subjects for the microfilaricidal and adulticidal effect of doxycycline showed significant reduction in microfilaraemia and antigenaemia after 8 weeks doxycycline treatment (8,9,10,11). It has been shown that doxycycline treated female worm are sterilized for long term even permanent sterilization and majority of worms (90% in LF) are died (4). The in vitro studies also demonstrated that antibiotics such as doxycycline, oxytetracycline and rifampicin have good activity against Wolbachia (12,13). Thus different studies conducted on anti-Wolbachia treatment in vivo (animal and human) and in vitro demonstrated lethal effects on filarial parasite viability. The present study aimed to investigate the microfilaricidal and macrofilaricidal effect of tetracycline/ doxycycline in vitro/ in vivo.

MATERIALS AND METHODS

In vitro anti-filarial studies

Microfilariae (mf) were obtained from the peritoneal cavity and adult worm collected from the lymphatic tissue of infected Mastomys natalensis. Microfilariae and adult worm were washed with RPMI 1640 medium, plated on sterile plastic Petri-plates and incubated at 37[degrees]C for 1 h to remove Mastomys natalensis peritoneal exudates cells. The mf were collected from Petri-plates, washed with RPMI-1640 medium and used for in vitro maintenance. The collected mf separated by nuclepore filtration.

The effect of tetracycline on motility of B. malayi mf and adult was carried out in RPMI 1640 medium containing 5% FCS, 2mM L-glutamine, 100[micro]g/ml streptomycin and 100U/ml penicillin. The mf (4000-5000 per well) and L3 (1-5 worms) were distributed in 6-well plate containing various concentrations of tetracycline (10 1/4g/ml, 20 1/4g/ ml, 50 1/4g/ml, 100 1/4g/ml) for mf and (20 1/4g/ ml, 50 1/4g/ml, 100 1/4g/ml) for L3 respectively in RPMI-1640 medium along with control (without tetracycline) and the motility was recorded at define time intervals for mf (upto 249h) and adults (upto 76h) after treatment.

In vivo anti-filarial studies

The study was carried out after taking information consent from filarial and healthy normal subjects residing in Raipur city, Chhattisgarh, India. A total of 14 microfilaraemic subjects (10 male and 4 female) age group of 14 to 62 were selected for the study. The range mf in 20 [micro]l blood sample in male was 8-17 and in female was 2-40 recorded. The study protocols as well as the use of biological samples from human subjects were approved by the Ethical committee of Jiwaji University, Gwalior, (M.P). The Doxycycline was distributed to selected subjects at a dose of 200 mg/day for 30 days. Before and after treatment a 20 [micro]l finger prick blood samples in duplicate were drawn from each subject between 20:00 to 22:00 hours. Thick smears were made and stained with Giemsa stain. The stained smears were examined under the microscope for mf count.

The adulticidal function of doxycycline was assessed by monitoring the adult movement of W. bancrofti in lymph nodes of various organs of the microfilaraemic subjects and one untreated filarial human subject, by Droppler pulse Sonography.

RESULTS AND DISCUSSION

In vitro effect of tetracycline

Though the chemotherapy with DEC is a comparatively a cheaper and cost effective but an important drawback associated with DEC is its side effects. The adverse effect of DEC limits its total acceptance by the population thus there is a need to find out safe treatment for LF. The antibiotic tetracycline/ doxycycline target the filarial endosymbiont, Wolbachia, opened new vistas for anti-filarial therapy. The microfilaricidal as well as macrofilaricidal function of tetracycline has been demonstrated in vitro (6) and in vivo in animal and human subjects (14).

Here, the impact of tetracycline exposure at different concentration on mf was studied in vitro and loss of mf motility was recorded as drug effectiveness on mf. The results indicated that the loss of motility was very high at concentration of 100[micro]g/ml and 50[micro]g/ml in comparison to 20[micro]g/ml, 10[micro]g/ml and control group [Table 1]. The motility was completely lost with increased exposure time (144h and 214h) in 100[micro]g/ml and 50[micro]g/ml concentration treated mf group. There was no notable difference in the motility loss in 10[micro]g/ml treated mf group and in control group. Therefore, it revealed that the drug tetracycline is highly microfilaricidal in nature and the effect was found dose dependent. Previous in vitro and in vivo Anti-Wolbachia treatment studies demonstrated the lethal effects of tetracycline on filarial parasite viability (2,3,11). Similarly the treatment with tetracycline effectively blocks embryo development in two filarial nematodes namely B. pahangi and D. immitis (13).

The in vitro effect of different tetracycline concentrations on adult worms was showed that loss of motility was higher in 100[micro]g/ml and 50[micro]g/ ml at 36h after treatment [Table 2]. The sex wise analysis showed that the motility was remaining higher (51-75%) in male and lower (26-50%) in female at 36h of 100 Mg/ml treatments. After 36 h the motility was gradually decreases in both male and female worms and is completely lost in female worms at 56h of exposure. But in male worms motility was remained up to 11-25% at 56h of exposure and remains the same at 76h of 100 Mg/ml treatments. As the concentration of drug decreases the motility was inhibited both male and female but intensity was very low. In comparison to male, the motility of female adult worms was completely lost after 56h at 50 [micro]g/ml tetracycline concentration. The loss of motility was efficiently higher in female adult worms than male adult worms at all tetracycline concentrations tested. It was indicated that the effect of tetracycline on adult filarial worms was more rapid in comparison to its effect on mf and it was gender dependent also. Thus tetracycline is an established antibiotic and is targeted to Wolbachia bacteria present within the filarial worms (15). The gender dependent difference in adult filaricidal effect of tetracycline could be attributed to the differences in Wolbachia load of the filarial worms. The findings of Foster et al (16) supported our observation that the female worms harbor much higher loads of Wolbachia in comparison to their male counter parts. The death of filarial worms following the elimination of Wolbachia indicates the inevitable association of these two organisms.

In vivo effect of Doxycycline

Thirty days course of doxycycline (200mg/day) was given to 10 male and 04 female human subjects with mf count 8-17 in male and 2-40 in female and was monitored upto 6 months after treatment. The mean mf count before treatment was 9.8 in male and 20 in female and the mf count decreased substantially in both the groups one month after the start of treatment [Table 3]. In male subjects infection was reduced gradually upto 6 months and it was observed after one month of treatment. Two out of 10 male subjects were found to be negative for mf count from 3rd month of treatment and remained negative during whole observation period (6 month). The gradual decrease in mf count was also observed in case of female subjects [Table 3]. After one month of treatment 14.75 mean mf count was recorded and which was reduced upto 5.5 mean mf count at 6 months after treatment. It was observed that doxycycline treatment effectively reduced the mf load due to targeting endosymbiont Wolbachia. These observations are supported by the reports published on killing of Wolbachia by doxycycline and subsequent reduction in microfilaraemia (6,13,15,17). A significant reduction as well as complete elimination of microfilaraemia at 12 months after 6 weeks doxycycline treatment was reported (8,10,11,18). The double blind field trail of doxycycline (200 mg/ day) for 8 week treatment on infected individuals, almost completely eliminate microfilaraemia, which was sustained for at least 8 to 14 months after treatment (10). The advantage of the study is that a shorter treatment time (4 weeks) provides a gradual reduction in the mf count. Treatment with doxycycline/ tetracycline proves a effective tool to check transmission of filariasis from one host to another by lowering the mf load in the peripheral blood of the vertebrate host.

ACKNOWLEDGMENTS

The authors thankful to Head, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur for providing facilities to carry out this study. Thanks also due to the Chairman, Ethics Committee, Jiwaji University, Gwalior for approving the ethical permission to undertake this study. Special thanks to Head, Department of Entomology, DRDE, Gwalior for valuable support.

REFERENCES

(1.) Ramaiah, K.D., Ottesen, E.A. Progress and impact of 13 years of the global programme to eliminate lymphatic filariasis on reducing the burden of filarial disease. PLoS Negl. Trop. Dis., 2014; 8: e3319.

(2.) Taylor, M.J., Hoerauf, A. Wolbachia bacteria of filarial nematodes. Parasitol. Tod., 1999; 15: 437-42.

(3.) Taylor, M. J., Hoerauf, A. A new approach to the treatment of Filariasis. Cur. Opin. Inf. Dis., 2001; 14: 727-31.

(4.) Hoerauf, A. Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr. Opin. Infect. Dis., 2008; 21: 673-681.

(5.) Hoerauf, A., Nissen-Pahle, K., Schmetz, C., Henkle-Duhrsen, K., Blaxter, M.L., et al. Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J. Clin. Inv, 1999; 103: 11-18.

(6.) Smith, H., Rajan, T.V. Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro. Exp. Parasitol., 2000; 95: 265-70.

(7.) Rao, R., Weil, G. J. In vitro effects of antibiotics on Brugia malayi worm survival and reproduction. J. Parasitol., 2002; 88(3):605-11.

(8.) Hoerauf, A., Mand, S., Volkmann, L., Buttner, M., Marfo-Debrekyei, Y., et al. Doxycycline in the treatment of human Onchocerciasis: Kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms. Micro. Infect., 2003; 5: 261-273.

(9.) Molyneux, D.H., Bradley, M., Hoerauf, A., Kyelem, D., Taylor, M.J. Mass drug treatment for lymphatic filariasis and onchocerciasis. Trends Parasitol., 2003; 19: 516-522.

(10.) Taylor, M.J., McKunde, W.H., McGarry, H.F., Turner, J. D., Mand, S., Hoerauf, A. Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomized placebo-controlled trial. Lancet, 2005; 365: 2116-21.

(11.) Taylor, M. J., Hoerauf, A., Bockarie, M. Lymphatic filariasis and onchocerciasis. Lancet, 2010; 376: 1175-1185.

(12.) Genchi, C., Sacchi, L., Bandi, C., Venco, L. Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion. Parasitologia., 1998; 40: 247-9.

(13.) Bandi, C., McCall, J.W., Genchi, C., Corona, S., Venco, L. et al. Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. Intern. J. Parasitol., 1999; 29: 357-64.

(14.) Srivastava, K., Bhattacharya, S. M. Tetracycline, a tool for transmission blocking of Brugia malayi in Mastomys coucha. Curr. Sc., 2003; 85: 588-89.

(15.) Hoerauf, A., Volkmann, L., Hamelmann, C., Adjei, O., Autenrieth, B., et al. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet., 2000; 355: 1242-3.

(16.) Foster, J., Ganatra, M., Kamal, I., Ware, J., Makavora, K., et al. The Wolbachia genome of Brugia malayi: endosymbionts evolution with in human pathogenic nematodes. PLoS Biology., 2005; 3(4): 599-613.

(17.) Hoerauf, A., S. Mand, O. Adjei, B. Fleischer, and D. W. Buttner. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet, 2001; 357: 1415-1416.

(18.) Tamarozzi, F., Halliday, A., Gentil, K., Hoerauf, A., Pearlman, E., Taylor, M.J. Onchocerciasis: the role of Wolbachia bacterial endosymbionts in parasite biology, disease pathogenesis, and treatment. Clin. Micro. Rev., 2011; 24: 459-468.

Subuhi Khan [1] *, Vanadana Dixit [1], Salauddin Qureshi [2], AK Gupta [3] and GBKS Prasad [1]

[1] School of Studies in Biochemistry, Jiwaji University, Gwalior - 474 011, India.

[2] IVRI, Izatnagar, India.

[3] School of Life Sciences, Pt. Ravishankar Shukla University, Raipur - 492 010, India.

http://dx.doi.org/10.22207/JPAM.11.4.48

(Received: 09 November 2017; accepted: 24 December 2017)

* To whom all correspondence should be addressed.

Tel.: +91-8941907072; E-mail: khansubuhi8@gmail.com
Table 1. Effect of tetracycline on the motility of microfilariae of
Brugia malayi

                        Period of exposure in hours

Tetracycline
conc. / ml     0h   36h   60h   84h   108h   120h   132h   144h

100[micro]g    4+   3+    2+    2+     1+     1+     1+     --
50[micro]g     4+   4+    4+    4+     3+     2+     2+     1+
20[micro]g     4+   4+    4+    4+     4+     4+     3+     3+
10 [micro]g    4+   4+    4+    4+     4+     4+     4+     4+
Control        4+   4+    4+    4+     4+     4+     4+     4+

                        Period of exposure in hours

Tetracycline
conc. / ml     156h   168h   180h   192h   214h   234h   249h

100[micro]g     --     --     --     --     --     --     --
50[micro]g      1+     1+     1+    +/-     --     --     --
20[micro]g      3+     3+     3+     3+     2+     2+     2+
10 [micro]g     4+     4+     4+     4+     3+     3+     3+
Control         4+     4+     4+     4+     4+     4+     3+

4+ = 76-100% motility, 3+ =51-75% motility, 2+ = 26-50 % motility, 1+
= 11-25% motility, +/-= 1-10% motility, -- =0 % motility

Table 2. Effect of tetracycline on motility of adult worms of
Brugia malayi

Tetracycline   Sex     Period of exposure in hours

conc. / ml             0 h   36h   56h   76h

100[micro]g    Male    4+    3+    1+    1+
              Female   4+    2+    --    --
50[micro]g     Male    4+    3+    1+    1+
              Female   4+    3+    +/-   --
20[micro]g     Male    4+    4+    3+    3+
              Female   4+    3+    1+    +/-
Control        Male    4+    4+    3+    3+
              Female   4+    4+    3+    3+

4+ = 76-100% motility 3+ =51-75% motility
2+ = 26-50 % motility 1+ = 11-25% motility

+/- = 1-10% motility --=0 % motility

Table 3. Effect of Doxycycline therapy on microfilaraemic subjects

Observations               Group     Before      After treatment
                                    treatment   1 month   2 month

No. Positive for mf         Male       10         10        10
                           Female      04         04        04
No. successfully treated    Male       10         10        10
                           Female      04         04        04
Completely cured            Male       00         00        00
                           Female      00         00        00
Mean Mf                     Male       9.8       3.14       1.5
                           Female      20        14.75     12.8

Observations                      After treatment

                           3 month   4 month   6 month

No. Positive for mf          08        08        08
                             04        04        04
No. successfully treated     10        10        10
                             04        04        04
Completely cured             02        02        02
                             00        00        00
Mean Mf                      0.3       0.3       0.3
                             7.3       7.5       5.5
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Author:Khan, Subuhi; Dixit, Vanadana; Qureshi, Salauddin; Gupta, A.K.; Prasad, G.B.K.S.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Dec 1, 2017
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