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 anserina isolated from

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 and is transmitted by a soft known as . 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 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). 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 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. Samples were incubated at 37°C, as The purified clones were processed for sequencing by using 3216 Afr. J. Microbiol. Res.

Table 1. Information of the sequences retrieved from the GenBank and used for the phylogenetic analysis in the current study. Representative of each subspecies was chosen for comparison.

Year of the Percentage similarity to that of S. No. Accession numbers Name of the organisms submission B. anserina of current study (%)

1 X75201.1 Borrelia anserina 1993 97

2 DQ849626.1 Borrelia anserina 2006 96

3 AY604979.1 Borrelia turicatae 2004 91

4 EF688579.1 Borrelia sp. 2007 91

5 DQ855534.1 Borrelia hermsii 2006 91

6 AY604980.1 Borrelia parkeri 2004 90

7 AY604981.1 Borrelia miyamotoi 2004 89

9 GU357619.1 Borrelia crocidurae 2009 88 10 GU357617.1 Borrelia duttonii 2009 88

11 DQ346831.1 Borrelia recurrentis 2005 88

BigDye® terminator sequencing kits (Applied Biosystems, Foster contrast microscope (Figure 1). City, CA), following the manufacturer's instructions. Briefly, the reaction contained 3 µl 5x sequencing buffer, 2 µl Big Dye, 1.8 µl primer (2 µM), 3 µl PCR product and 10.2 µl of PCR quality water. The following thermo-profile was used for the sequencing reaction Confirmation of spirochaetes of 25 cycles at 96°C for 4 min; and at 96°C for 15 s, 50°C for 10 s and 60°C for 4 min. Then the reactions were precipitated with 3 M All the 40 samples which showed granular growth were sodium acetate and 95% ethanol. Subsequently, the samples were processed for indirect immunoflourescence assay. centrifuged and pellets were washed with 75% ethanol and dried at Despite using the polyclonal IgG Borrelia antibodies all room temperature. Finally, the pellet was reconstituted in formamide and load to DNA sequencer (Applied Biosystems, Foster the samples showed clear fluorescence. Furthermore City, CA). these samples were subjected to PCR for molecular identification and genetic characterization.

Sequence and phylogenetic analysis Molecular and phylogenetic analysis To generate consensus sequence, the raw sequences were pair- wise aligned using SeqMan programme in Lasergene DNASTAR (Madison, USA). Phylogenetic analysis was performed using the DNA was extracted from the spirochaetal culture and sequences from current study and from the GenBank (accession subjected to the PCR for the amplification of the flagellin numbers are presented in Table 1), characterized previously. gene, 32 (80%) out of 40 samples amplified a product of Bayesian analysis was carried out using MR Bayes 3.1.2 with two 750 bp (Figure 2). The products were sequenced (n=5) millions generation of the two replicates. The phylogenic estimates after their topo-cloning. BLAST analysis indicated that the were performed by Neighbor-Joining 1000 bootstrap replicates. The posterior probabilities of Bayesian were calculated with the isolate having accession number JF693808 was found consensus of the trees and burn in trees was not included in the highest percentage similarity to the B. anserina analysis. sequences present in the GenBank (shown in Table 1). The phylogenetic analysis indicated that all the species of Borrelia could be well differentiated from each other. We RESULTS have observed that sequence in our study clustered close enough with anserina subspecies to be declared it as B. Isolation of spirochaetes anserina. The high bootstrap values indicated the confidence. Since there were just two sequences of B. The BSK-H complete medium inoculated with tick anserina available, it was hard to link to the variability samples becomes turbid on the 7th day post inoculation among these isolates. Based on the overall topology of in 40 (18%) samples. Best growth was observed at 37°C the tree it is evident that all the Borrelia species except B. after 7th day of inoculation. Samples showed granular miyamotoi, B. crocidurae, B. duttonii are variable groups growth earlier than the other; this is perhaps due to the of B. anserina (Figure 3). The sequence alignment of presence of the infection inside the tick sample. Initially, flagellin protein to that of previously characterized 2-3 passages took 18-20 days for the isolation of Borrelia sequences from representative subspecies indicated the in some ticks, after that the passage time was observed genetic difference at the amino acid level (Figure 4). At about 5-7 days for all the isolates. Numerous viable first glance, this alignment demonstrated that there have spirochaetes were observed under dark field and phase been several silent mutations in the flagellin gene since Aslam et al. 3217

Figure 1. Phase contrast microscopic image of B. anserina.

Figure 2. PCR amplified products (750 bp) for flagellin gene (flaB) of B. anserina. M=marker, positive control and positive samples are mentioned. 3218 Afr. J. Microbiol. Res.

B.hermsii/USA/2006 (DQ855535) B.hermsii/USA/2004 (AY597790) 96 B.hermsii/USA/1990 (X53940) B.hermsii/Spain/2009 (GU357620) 73 B.hermsii/USA/2004 (AY597789) B.hermsii/USA/2004 (AY597794) B.hermsii/USA/2004 (AY597792) 78 85 B.hermsii/USA/2006 (DQ855531) 66 B.hermsii/USA/2004 (AY597793) B. hermsii B.hermsii/USA/2009 (GQ175063) 100 100 B.hermsii/USA/2009 (GQ175059) B.hermsii/USA/2004 (AY597806) B.hermsii/USA/2004 (AY597801) B.hermsii/USA/2004 (AY597799) 57 99 B.hermsii/USA/2006 (DQ855534) B.hermsii/USA/2007 (EU194839) B.hermsii/USA/2004 B.coriaceae/Japan/1995 (D82864) B. coriaceae 41 B.parkeri/USA/2004 (AY604980) 87 B.parkeri/USA/2005 (AY934623) 99 B.parkeri/Japan/1995 (D82863) B. parkeri B.parkeri/USA/2000(AF307102) 88 B.parkeri/USA/(AF307101) 99 B.texasensis/USA/2000 (AF264901) B. texasensis 92 Borrelia sp./USA/2008 (EU492387) Uncharacterized Borrelia spp 43 Borrelia sp./USA/2007 (EF688579) B.turicatae/Florida/2005 (AY934630) 27 B.turicatae/Japan/1995 (D82862) 89 B. turicatae B.turicatae/America/2004 (AY604979) 66 B.turicatae/Florida/2005 (AY934629) B.anserina/PL/2006 (DQ849626)

100 B.anserina/Sweden/93 (X75201) B. anserina 69 B.anserina/Pakistan/2009 (JF693808) 100 B.miyamotoi/USA/2004 (AY604981) B. miyamotoi B.miyamotoi/Poland/2009 (FJ874925) B.crocidurae/Spain/2009 (GU357619) B. crocidurae 53 B.duttonii/Spain/2009 (GU357617) 100 B.duttonii/Japan/1995 (D82859) 99 65 B.duttonii/UK/2005 (DQ346837) 31 B.duttonii/UK/2005 (DQ346835) 30 B. duttonii and B. recurrentis B.duttonii/Spin/2009 (GU357618) 36 B.recurrentis/UK/2005 (DQ346830) B.recurrentis/UK/2005 (DQ346831) 65 B.recurrentis/UK/2005 (DQ346831)(2)

0.01

Figure 3. Phylogenetic tree for the flagellin gene (fla B) of B. anserina. Figure shows phylogenetic tree constructed by Neighbor-Joining method on the flagellin gene. B. anserina/Pakistan/2009 is highlighted with a black filled circle (). Numbers indicate the bootstrap values (1000). Horizontal distances are proportional to sequence distances. Sequences are named with species/country of origin/year of isolation and (accession number).

the difference in the amino-acid is not high compared to the world (Lisboa et al., 2009). A study conducted by nucleotide sequence of the gene. Bhatti (1989) showed that incidence rate of spirochaetosis was around 10% in Pakistani poultry and this has been observed over a period of nine years. A DISCUSSION study exposed the vector role of A. persicus, which revealed that it transmits a number of bacteria which Due to fowl spirochaetosis, caused by B. anserina, includes Staphylococcus aureus, Salmonella pullorum poultry industry faces heavy economic losses throughout and Escherichia coli, B. anserina (Shah et al., 2006). In Aslam et al. 3219

Figure 4. An alignment for the flagellin protein of Borrelia isolates. Putative amino acid sequence of flagellin protein of B. anserina/Pakistan/2009 was aligned with the flagellin protein of each representative of subspecies. B. anserina/Pakistan/2009 is marked with black circle ().

Pakistan, the vector role of A. persicus and A. resistant to nalidixic acid leading to pure growth. We used vespertilionis in the transmission of spirochaetosis in 100 µg/ml nalidixic acid (Negrom®) to avoid the layer chicken was already confirmed through the isolation contamination of different microorganisms, which can of B. anserina from naturally infected ticks and their contaminate the spirochaetal growth. These findings are experimental transmission (Shah et al., 2004). As in the in accordance to the Barbour 1984 isolated the Borrelia present study we used Barbour Stoenner Kelly (BSK-H) very first time by using BSK medium. complete medium for the isolation of the spirochaetes Isolation rate of Borrelia up to 9% from tick samples from different tick samples, this complete medium (Sigma has been reported by Davidson et al. (1999) whereas in USA) is used frequently by the scientists (Ataliba et al., the present study an isolation rate of 18% has been 2007). In current report, a 40 (18%) out of 220 ticks demonstrated from Argas ticks. It has previously been samples showed the viable spirochaetes. Results of the observed that tick infestation is higher in summer, in present study showed higher isolation rate as compared younger birds and in areas having high population of to the isolation rate of Borrelia during 1997 was reported poultry (Shah et al., 2004). as 8.5% of local ticks which was astonishing at that time Microscopy found to be difficult due to the obvious in view of the previous difficulties in isolation of this reason of motility of the spirochaetes, for microscopy organism (Nuttall et al., 1994). Whereas BSK-H medium culture was kept at 4°C it slow-down the movement of the was used with micro-aerophilic conditions though Borrelia. This practice was very helpful to observe Livesley et al. (1994) found the best growth in these spirochaetes in the microscopic field. Additional conditions. None of the cultures was observed confirmation for the identity of the isolates as B. anserina contaminated, as it was confirmed by incubation of the was established by indirect immunoflourescence, a culture having the granular growth of Borrelia at 37°C for method used in the early detection of B. burgdorferi 24 h on solid media. We also found that Borrelia is (Steere et al., 1983). All positive isolates reacted to 3220 Afr. J. Microbiol. Res.

polyclonal IgG anti Borrelia antibodies but no To our knowledge, this is first study conducted in immunoflourescence was detected with negative control. Pakistan regarding the genetic analysis of B. anserina, The multiplicity of Borrelia genus is attracting a lot of the causative agent fowl spirochaetosis. This study will scientific curiosity regarding microbial evolution. To raise the importance of the spirochaetes as one of the understand the adaptation of the Borrelia genus to major problem in poultry industry and set off the vectors and vertebrate reservoirs, the environment for the deeper studies into this subject. phylogenetic analysis of Borrelia species will be essential Understanding the genetic background of circulating B. to identify pathogenic organisms. Scientists used three anserina in Pakistan will help the pharmaceutical sector different genes like 16s rRNA (Marconi and Garon, 1992) while designing vaccine and therapeutics. outer surface protein A (OspA) Wallich et al. (1992) and flagellin (Picken, 1992) genes as phylogenetic markers. Reason to focus on the flagellin gene was that it encodes ACKNOWLEDGEMENTS the endoflagellar protein and its diversity for characterization of the Borrelia is now well accepted Authors want to extend their gratitude to Higher (Fukunaga et al., 1996). Most of the phylogenetic Education Commission of Pakistan (HEC) for funds and analysis has been conducted based of fla B gene. We they thank the personnel of the Department of have also shown that all the species were clearly Bacteriology, National Veterinary Institute, Uppsala, differentiated from each other; B. anserina has a Sweden, and Alan G. Barbour for providing positive similarity of 91 and 88% to B. hermsii and B. crocidurae, control. respectively. Results of phylogenetic analysis of the present study are in accordance to the results of Noppa REFERENCES et al. (1995). 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