Wolbachia have established a mutualistic association with filarial nematodes and has a phenomenal implication in its normal development, reproduction and survival. Elimination of Wolbachia by tetracycline class of antibiotic compounds have been suggested and successfully implemented for the treatment of lymphatic filarial parasites. Thereby, is necessary to assess the prevalence of the Wolbachia in B. malayi before such new strategies are employed, across the world. In the present communication, the presence of Wolbachia and phylogenetic affiliation in B. malayi collected from Sevagram, Maharashtra, India, has been addressed

Prakash, B.M.

Puttaraju, H.P.

Ravi Kumar, H.

Sampath Kumar, S.

ePrints@Bangalore University

Vol 5|Issue 2| 2015 | 96-98.  96 | P a g e          e-ISSN 2248 - 9169    Print ISSN 2248 - 9150  International Journal of Experimental Pharmacology  www.ijepjournal.com  WOLBACHIA ASSOCIATION AND ITS PHYLOGENETIC AFFILIATION OF BRUGIA MALAYI PARASITES FROM INDIA  Ravi kumar H*, Prakash BM, Sampath Kumar S and Puttaraju HP  Department of Biological Sciences, School of Natural Science, Jnanabharathi Campus, Bangalore University,  Bangalore- 560056, India.           INTRODUCTION Filarial nematodes like Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause several important human diseases across tropics and subtropics. They belong to the order Spirurida and family Onchocercidae, have been reported to harbour the Wolbachia endosymbionts [1]. These bacteria have been implicated not only in establishing a mutualistic association with filarial nematodes but also in the pathogenesis of filarial disease [2]. Wolbachia acts on the host immune system and accelerates the rate of inflammation. During pathogenesis the β cell proliferation of the host is directed specifically towards Wolbachia surface antigens which strengthen the possible role of Wolbachia in filarial pathogenesis [3]. After the death of the nematodes, the host respond to Wolbachia by releasing stimulatory and modulatory factors from neutrophils and monocytes [4].   The typical immunological response of the host in producing mimics of lipopolysaccharide and activation of Toll like receptor-4 (TLR4) are known  to  be  induced  by   Corresponding Author  Ravi Kumar H Email id: ravikumarh79@gmail.com  Wolbachia [6]. The regulation of Th1 and Th2 cytokines, which are the potential targets for filarial pathogenesis, is known to be governed by TLR4 [7]. The strategies of combating filarial nematodes through Wolbachia by tetracycline class of antibiotic compounds directly hinder their proliferation and manage their pathogenesis [8]. In view of the importance of Wolbachia for the survival, developmental stages, reproduction and pathogenesis, it is necessary to assess the prevalence of Wolbachia in B. malayi from different geographical locations in India. However, earlier research by Hoti, et al., [9] and Gayen, et al., [10] identifies the occurrence of Wolbachia only in W. bancrofti collected from various geographical locations in India. The present study is an attempt to screen for the presence of Wolbachia in B. malayi (Bm-I) collected from the Jamnalal Bajaj Tropical Disease Research Centre at Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra, India and to fill this impending gap.  MATERIALS AND METHODS  Genomic DNA was extracted from microfilariae (approximately 1500 in number) by Column based Animal tissue kit (Chromous BiotechTM Pvt, Ltd, Bangalore, India) with manufacturer’s protocol. DNA was quantified through ABSTRACT  Wolbachia have established a mutualistic association with filarial nematodes and has a phenomenal implication in its normal development, reproduction and survival. Elimination of Wolbachia by tetracycline class of antibiotic compounds have been suggested and successfully implemented for the treatment of lymphatic filarial parasites. Thereby, is necessary to assess the prevalence of the Wolbachia in B. malayi before such new strategies are employed, across the world. In the present communication, the presence of Wolbachia and phylogenetic affiliation in B. malayi collected from Sevagram, Maharashtra, India, has been addressed.  Keywords: Wolbachia, Lymphatic filarial parasites.  Vol 5|Issue 2| 2015 | 96-98.  97 | P a g e   Bio-photometer (Eppendorf AG, Hamburg, Germany). A polymerase chain reaction (PCR) assay was done through thermocycler (Eppendorf AG, Hamburg, Germany), with the reaction mixture containing 10 μl 10X buffer (5 Prime Eppendorf), 3 μl 25 mM MgCl2, 1.25 μl dNTPs (10 mM each), 1 μl 10 pmoles of both forward and reverse primers, 1.5 unit of Taq DNA polymerase(5 Prime Eppendorf) and template DNA. The PCR conditions followed for each step included 3 min at 950C for the initial denaturation step followed by 35 cycles of 45 s at 940C (denaturation), 1 min at 510C (annealing), 1min at 720C ( primer extension) and 7 min at 720C for the final extension. For amplication filarial-specific 28 S rRNA, Wolbachia 16 S rRNA and Wolbachia surface protein (wsp) gene primers [9,10,11]. PCR products were resolved in 1.2% agarose gel and stained with green view dye (Chromous BiotechTM Pvt, Ltd, Bangalore, India). The amplicons were observed and recorded in a gel documentation unit (Alpha Imager (R) EP, Canada).The size of the PCR product was determined using a 1-kb ladder (GeNeiTM, Bangalore, India).  The PCR product of wsp gene were purified using Chromous PCR Clean-up kit (Chromous BiotechTM, Bangalore, India) and directly sequenced with respective primers using an automated sequencer (3130 Genetic Analyzer, ABI, Foster city, California, USA). The sequences obtained have been deposited in GenBank under the accession number JX506736. Phylogenetic analysis of the wsp gene sequence was done at BLAST-x program at NCBI. Multiple sequence alignment was done by using CLUSTAL W program. The phylogenetic trees were constructed using Kimura-2-distances and the Neighbor-Joining algorithm was computed using MEGA 4 program [12].   Fig. 1. PCR amplification of B. malayi (Bm-I) using filarial 28S rRNA-specific primers (Lane 1), Wolbachia- specific 16S rRNA primers (Lane 2), Wolbachia surface protein-specific primers (Lane 3) and Lane M is the migration pattern of the 1 kd DNA ladder.  Fig. 2. Phylogenetic tree of Wolbachia based on the wsp sequences, constructed from Kimura-2-distance and the Neighbour-Joining algorithm. The numbers near the node indicate percentage of 1000 bootstrap replicates. Names correspond to host species. The GenBank accession numbers are also mentioned.   RESULTS AND DISCUSSION  PCR amplification was carried out with Wolbachia- specific 16S rRNA and wsp gene primers to confirm the presence of Wolbachia infection in B. malayi. 16S rRNA gene amplified at 207 bp and wsp gene amplified around 590 bp fragments, whereas filarial-specific 28S rRNA primers was used confirming the quality of template DNA and authenticity of the experimental protocol yielded distinct band at 150 bp as shown in the figure-1.   The evolutionary history of Wolbachia lineages in the B. malayi of Indian populations was investigated by phylogenetic analysis performed with Neighbour-Joining algorithm using Kimura-2-distance. Direct sequencing of the PCR products gave only one sequence without double peaks, indicating the presence of only one strain in the Bm-I. These sequences have been submitted to the Genbank    database and phylogenetic tree based wsp gene was constructed. B. malayi grouped in D supergroup as shown in the figure 2. The wsp gene sequences of Wolbachia from Bm-I strain showed highly homology with the previously reported B. malayi strain Bm-1, proving they belonged to the same strain.  The study investigated the presence and phylogenetic affiliation of Wolbachia in B. malayi (Bm-I) from India. The presence of Wolbachia in W. Bancrofti has been well documented in India by Hoti et al [8] and Gayen et al [9]. B. malayi is considered to be high endemic to several states of India since decades. But there is a lack of information on Indian strain Bm-I. Thus, the current preliminary study has shown that Wolbachia is present in B. malayi (Bm-I) and the phylogenetic analysis shows the sequence similarity with members of Bm-1 strain of D Vol 5|Issue 2| 2015 | 96-98.  98 | P a g e   super group and correlated well with the earlier works of Bazzocchi et al [11] and Casiraghi et al [1]. Wolbachia has displayed a mutualistic relationship with nematodes, and therefore elimination of Wolbachia by tetracycline class of antibiotic compounds decreases the host fitness and ultimately leads to its mortality [10]. The prevalence of Wolbachia in B. malayi from various geographical areas across India, and the extent of mutualism, its application in filarial  management    programmes    is    an   interesting proposition for future studies.     ACKNOWLEDGEMENT Authors would like to thank DR. M. V. R. Reddy Dept. of Biochemistry, Jamnalal Bajaj Tropical Disease Research Centre at Mahatma Gandhi Institute of Medical Sciences, Sevagram, Maharashtra, India for providing B. malayi sample and highly thankful to the ICMR (No.5/8-7(309)V-2011/ECD-II, Vector Science Forum) for Research Associate position.  CONFLICT OF INTEREST The authors declare that they have no conflict of interest. REFERENCES 1. Casiraghi M, Anderson TJ, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: Comparison with the phylogeny of Wolbachia endosymbionts. Parasitology, 122, 2001, 93–103. 2. Bandi, C, Trees S, Brattig N. Wolbachia in filarial nematodes: Evolutionary aspects and implications for the pathogenesis and treatment of filarial disease. Veterinary Parasitology, 98, 2001, 215–238. 3. Lamb TJ, Le Goff L, Kurniawan A, Guiliano DB, Fenn K , Blaxter ML, Read AF, Allen JE. (2004). Most of the response elicited against Wolbachia surface protein in filarial nematode infection is due to the infective larval stage. Journal of Infectious Diseases, 189, 2004, 120–127. 4. Hise AG, Gillette-Ferguson I, Pearlman E. The role of endosymbiotic Wolbachia bacteria in filarial disease. Cell Microbiology, 6 (2), 2004, 97–104. 5. Taylor MJ, Cross HF, Bilo K. Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharide like activity from endosymbiotic Wolbachia bacteria. Journal of Experimental Medicine, 191, 2000, 1429–1436. 6. Andre A, Blackwell NM, Hall LR, Hoerauf A, Brattig NW, Volkmann L, Taylor MJ, Ford L, Hise AG, Lass JH, Diaconu E, Pearlman E. The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science, 295, 2002, 1892–1895. 7. Taylor MJ, Bandi C, Hoerauf AM, Lazdins J. Wolbachia bacteria of filarial nematodes: A target for control?. Parasitology Today, 16, 2000, 179–180. 8. Hoti SL, Sridhar A, Das PK. Presence of Wolbachia endosymbionts in microfilariae of Wuchereria bancrofti (Spirurida: Onchocercidae) from different geographical regions in India. Memórias do Instituto Oswaldo Cruz, 98(8), 2003, 1017-1019. 9. Gayen P, Maitra S, Datta S, Babu SP. Evidence for Wolbachia symbiosis in microfilariae of Wuchereria bancrofti from West Bengal, India. Journal of Bioscience, 35(1), 2010, 73-7. 10. Smith HL, Rajan TV. Tetracycline inhibits development of the infective-stage larva of ﬁlarial nematodes in vitro. Experimental Parasitology, 95, 2000, 265–270. 11. Bazzocchi C, Jamnongluk W, O’Neill S, Anderson TJC, Genchim C, Bandi C. Wsp gene sequences from the Wolbachia of ﬁlarial nematodes. Cell Microbiology, 41, 2000, 96–100. 12. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 2007, 1596-1599.  

Wolbachia Association and its Phylogenetic Affiliation of Brugia Malayi Parasites from India

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Wolbachia Association and its Phylogenetic Affiliation of Brugia Malayi Parasites from India

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