Flagellin Gene Based Phylogenetic Analysis of Pakistani Strain of Borrelia Anserina Isolated from Argas Ticks
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African Journal of Microbiology Research Vol. 6(13), pp. 3214-3221, 9 April, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1591 ISSN 1996-0808 ©2012 Academic Journals Full Length Research Paper Flagellin gene based phylogenetic analysis of Pakistani strain of Borrelia anserina isolated from Argas ticks Bilal Aslam, Iftikhar Hussain, Muhammad Shahid Mahmood*, Sajjad-ur-Rahman and Abu Baker Siddique Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan. Accepted 13 January, 2012 Fowl spirochaetosis is an acute septicemic disease of avian species caused by Borrelia anserina and is transmitted by a soft tick known as Argas persicus. In this study, the Argas ticks were collected for the isolation and characterization of the B. anserina. Of the 220 tick samples, 40 (18%) showed granular growth in BSK-H medium. Morphological examination was performed by using the dark field and phase contrast microscope. Furthermore, the entire batch of 40 samples reacted positively to polyclonal IgG anti-Borrelia sera in indirect immunofluorescent assay. For molecular characterization of the isolates, a polymerase chain reaction (PCR) was applied using gene specific primers for flagellin gene (fla B). This PCR amplified a product of 750 bp from the bacteria of 32 (80%) out of 40 positive samples. The fla B gene from five samples were sequenced; Sequence and phylogenetic analysis indicated that these isolates belong to B. anserina and clustered apart from other species of Borrelia. This is the first report presenting the phylogenetic analysis of B. anserina from Pakistan, despite the fact that it remained one of the most devastating diseases for poultry in the region. The results of this study shed light on the Borrelia population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology and nature of the bacterium. Such understanding is essential in an effort to control the number and impact of outbreaks that are occurring in poultry industry. Key words: B. anserina, spirochaetosis, poultry diseases, Pakistan, soft ticks. INTRODUCTION Poultry industry is the second largest industry in Pakistan bacterial structure, it acquires poor staining and is usually and is facing enormous problems regarding infectious diagnosed by dark field microscope and/or phase con- agents, which are causing heavy economic losses. Fowl trast microscope (Ataliba et al., 2007). The B. anserina spirochaetosis or avian borreliosis is one of the major carries two membranes; an outer membrane encloses tribulations; it is an acute septicemic disease of avian the protoplasmic cylinder while internal components of species. In Pakistan, the most prevalent genus of the soft cell are enclosed by inner membrane. B. anserina tick is Argas, a vector for avian borreliosis (Shah et al., contains sub-terminally attached 7-11 flagella for its 2006). The incidence of this disease is reported to be movement (Goldstein et al., 1994). These flagella are higher during May-August, which is mainly contributed by crucial for bacterial survival, whereas mutants lacking vector activation in summer (Chawla and Singh, 1969). flagella become rod shaped without wavelike structure Borrelia anserina is the causative agent of fowl (Motaleb et al., 2000). spirochaetosis. It is transmitted through Argas persicus Flagellar apparatus present in all spirocheates is a cell and is distributed globally. Morphologically, it is 0.2-0.5 structure that is common in all other motile bacteria µm in diameter and 10-50 mm in length. Due to complex (Noppa et al., 1995; Charon et al., 1992). Spirochaetes have the periplasmic flagella because they are situated in the periplasm. Unlike other spirochaetes the flagella of borreliae are not surrounded by an outer sheath layer *Corresponding author. E-mail: [email protected]. (Barbour et al., 1986). Flagellin is one of the Aslam et al. 3215 immunodominant antigens of Borrelia during infection recommended Barbour et al. (1984). After incubation granular because it is found that antibodies against the flagellin growth without cloudiness was observed in samples and was appear early as compared to the other antigens during confirmed by dark field and phase contrast microscopy for the presence of spirochaetes. Motility of Borrelia was noticed and the infection (Aslam et al., 2011). For this reason, additionally, indirect immunoflourescence assay was performed for scientists used flagellin gene for phylogenetic analysis in the confirmation of the isolates. Borrelia extensively with the purpose of developing diagnostic tools for Borrelia infection (Rosa et al., 1991). Sequence analysis of cloned genes encoding the flagellin Indirect immuno-flouresencent assay polypeptides, flaA and flaB, of several spirochaetes, Indirect immuno-flouresencent assay was performed as described (Koopman et al., 1993) revealed that products of flaA before Horta et al. (2004). Briefly, medium carrying spirocheates unlike flagellins of other eubacteria compose the flagellar were centrifuged at 12,000 × g for 10 min. The pellet was washed in sheath layer. No sequence homology was found in these 0.1 M phosphate-buffered saline (PBS). After an additional flagellins with eubacteria. However, flaB genes deficient centrifugation, pellet was again resuspended in PBS containing 1% in a signal peptide showed homologies with the flagellin bovine calf serum. A 10 µl of sample was dispensed into all the well of microscopic slide chamber except the control well. The slides gene of different motile bacteria (Parales and Greenberg, were air-dried and fixed in acetone for 10 min. A 10 µl of specific 1993). Borrelia IgG polyclonal antibodies (BactraceTM) were added to each Three different types of bacterial genes, such as 16s well of the chamber. The slides were incubated at 37°C for 30 min. rRNA (Marconi and Garon, 1992), outer surface protein A The slides were rinsed once, and then washed twice for 10 min per (OspA) and flagellin genes (Picken, 1992), have been wash in PBS. The slides were incubated with fluorescein-labeled proposed to be used as phylogenetic markers. The anti Borrelia IgG and washed as described earlier. The slides were mounted with gel mount under cover slips. Finally, slides were flagellin gene encodes the endoflagellar protein specific visualized under standard immunoflourescence microscope (Nikon) to spirochetes and due to its diversity it has extensively using suitable filters. been used for the identification of the Borrelia species. The endoflagellum is necessary for the movement of the spirochaetes and consists of one flagellin protein, which Flagellin gene amplification has a molecular mass ranging from 38-41 kDa. A The DNA was extracted from the spirochaetal culture using the conserved gene of 1-kb length encodes the flagellin DNeasy Tissue kit (Qiagen) in the BioRobot EZ1. Briefly, 5 ml of protein independently and is located on megabase linear culture was centrifuged at 5000 rpm and supernatant was chromosomes (Fukunaga et al., 1996). discarded followed by pallet resuspension into 200 µl of G2 buffer B. anserina is still prevalent throughout the world. (Qiagen). A 20 µl lysozyme was added and incubated at 37°C for Despite the fact that fowl spirochaetosis is one of the 30 min. Hereafter, EZ1 BioRobot procedure was followed for the DNA extraction. For the molecular identification of isolates, major constraints for poultry industry in Pakistan, some polymerase chain reaction (PCR) was performed using the specific attempts have been made on pathological aspects of primers for flagellin gene (flaB) with following sequences, FP: 5- spirochaetosis Hafeez (1979), but no work has been ACA TAT TCA GAT GCA GAC AGA GGT-3´, RP: 5´-GCA ATC conducted on the causative agent. This is the first study ATA GCC ATT GCA GAT TGT-3´ (Barbour et al., 1996). A total of 3 of its nature, which deal with genetic characterization µl DNA was used as template for the flagellin gene amplification in phylogenetic analysis of spirochaete B. anserina 25 µl of final volume of PCR master mix. Specifically, final volume of PCR master mix contained 2.4 µl of 25 mM of MgCl2, 0.6 µl of 10 circulating in poultry industry of Pakistan. nM of dNTPs, 0.6 µl of 10 µM of forward and reverse primers and 1 U of platinum Taq DNA polymerase (Invitrogen). Reaction was performed for 30 repeated cycles with the following profile: 94°C for MATERIALS AND METHODS 30 s, 52°C for 30 s and 72°C for 1 min, final extension at 72°C for 10 min and finally reaction was terminated at 4°C. A 0.8% Agarose Collection of Argas ticks gel electrophoresis with positive control was performed to visualize the positive PCR reaction under Gel documentation system Ticks (n=220) were collected throughout the summer season from (Dolphin- Doc, WEALTEC, USA). April to September 2008-2009 from different poultry farms at Faisalabad and Kmalia districts in Punjab Pakistan. A complete historical and clinical data of fowl spirochaetosis was recorded. Topo cloning of the PCR products Ticks were collected with the help of forceps and after collection ticks were saved into screw-capped plastic tubes having small The amplified PCR products were topo cloned using pCR 3.1 cotton swabs moistened with 1% mycostatin solution, to avoid TOPO cloning kit (Invitrogen). Final volume of 6 µl was used for the dehydration, and ticks were stored at 5°C until further use (Guner et reaction having 3 µl of PCR product, 2 µl of sterile water and 1µl of al., 2003). TOPO vector. The vectors with cloned product were propagated in E. coli (DH5α strain) cells and were then purified using Miniprep kit (Promega). Colony PCR was performed using flaB gene specific Spirochaete cultivation primers to confirm of the positive colonies. For the cultivation of B. anserina, a total of 220 dissected Argas ticks were inoculated into 5 ml of BSK-complete medium (Sigma- Sequencing Product No. B8291, USA) enriched with sodium bicarbonate and 6% rabbit serum.