The role of obligate endosymbiont Wolbachia in the pathogenesis and immune response to dog heartworm disease is well documented. In the present study, we explored some of the molecular characterization and phylogenetic study of endosymbiont-Wolbachia of canine heartworm (Dirofilaria immitis) from Northeastern state of India. Understanding the role of this endosymbiont as specific biomarker for presence of canine heartworm, a molecular characterization and study the phylogenetic relationship among other isolates from different parts of the world is carried out using FtsZ gene. The nucleotide sequence was submitted to GenBank and accession number obtained (JX524742). The percent identity among the Wolbachia endosymbiont in D.immitis ranges from 99.4 to 99.8 per cent. Phylogenetic characterization showed close identity with Wolbachia endosymbiont of USA isolate.
Wolbachia are common and widespread group of organisms found in reproductive tissues of arthropods and most of the filarial nematodes. These intracellular organisms were first observed within filarial nematodes in the early 1970’s by transmission electron microscopy (Kozek, 1977). It was only recently these organisms were identified as belong to the genus Wolbachia (Rickettsiales) – a gram negative bacteria. These gram negative bacteria are found in 100 per cent of the Dirofilaria immitis population and present in all the developmental stages of this filarial worm (Sironi et al., 1995). Current information on the distribution and phylogeny of Wolbachia in filarial nematodes suggests that Wolbachia is necessary for the host nematode. The reasons why Wolbachia is essential for filarial worm survival are due to (i) the evolution of the bacteria match that of the filarial worms, and phylogenic analysis have shown that the two organisms have been walking hand in hand for millions of years, (ii) the bacteria are transmitted from female to offspring and in this way Wolbachia bacteria increases its own fitness by increasing fitness of the host that is involved in its transmission and (iii) removal of Wolbachia (antibiotics/radiation) leads to sterility of female worms and eventual death of adults. It has been suggested that, while the filarial worm likely supplies the bacteria with amino acids necessary for growth and replication, Wolbachia on the other hand may produce several important molecules that are essential for heartworms, like glutathione and haeme (Foster et al., 2005).
In India association of Wolbachia with Wuchereria bancrofti from different geographical regions have been reported (Hoti et al., 2003; Gayen et al., 2010). NE region of India is established endemic for canine dirofilariosis (Borthakur et al., 2006; Borthakur et al., 2011; Borthakur et al., 2015; Bhattarcharjee and Sarmah, 2014). Though association of Wolbachia with D. immitis is reported from this part of India (Borthakur et al., 2013), the detail of molecular characterization is yet to ascertain. Here, we explored some of the molecular characterization and phylogeny of Wolbachia endosymbiont of D. immitis from Northeastern state of India.
Materials and Methods
The study was undertaken in two cities of North Eastern States of India viz. Guwahati, a city of Assam is located at the latitude of 26°11’0″ N and longitude of 91°44’0″ E having annual rainfall of 1500-2600 mm with an average altitude of 52 mts msl. Guwahati has a humid subtropical climate, falling just short of a tropical savanna climate. Aizawl, the capital city of Mizoram State of north east located at 23°43’27” N and 92°43’2″ E having annual rainfall of 2400-2962 mm with an average altitude of 1132 mts msl. In the summer the temperature ranges from 20–30 °C (68–86 °F), and in the winter 11–21°C (52–70°F). Both the cities are separated by surface distance of 550km. These states are endemic for D. immitis infection in canine. Dirofilaria immitis adult worms were collected from the heart of infected dogs (n=25) at post mortem and transported to the laboratory in physiological saline from Aizawl and Guwahati cities. All worms were identified as D.immitis as per their predilection site and on the basis of morphology of worms (Soulsby, 1982) .These worms were washed several times in physiological saline and store in 70% ethanol at -200C till DNA extraction. Approximately 5mL of blood was drawn from the cephalic vein from clinically confirmed heartworm infected dogs (n=25) in disodium salt of ethylene diamine tetraacetic acid (Na2EDTA) vacuum tubes. Subsequently, microfilarial identification was done. The immunological diagnosis was based on the presence of heartworm antigens in tested blood samples and was performed with a commercially available ELISA test kit (SNAP®4Dx) following manufacture’s test protocol. Molecular evidence was based on amplification of worm targeted DNA.
The adult (female) worms were process for DNA isolation by using a commercial DNA extraction kit (QIAGEN® Catalogue No. 69504). Briefly, about 1 cm of parasite section was triturate in tissue homogenizer in 200 µl of AL buffer along with 20 µl of Proteinase K. The homogenate is then incubated at 56°C for 10 min and added with 200 µl absolute alcohol and vortexed. The homogenate is applied to spin column and spun at 6000 x g. The spin column binded with DNA is then washed with 500 µl of wash buffer – AW1 and spun again. This is followed by addition of 500 µl of AW2 and centrifuge at 20, 000 x g followed by subsequent elution in nuclease free water. The eluted purified DNA is then stored at -20 °C till further use. Isolation of genomic DNA from positive blood was also carried out using the DNeasy Blood and Tissue kit (Qiagen® Kit, Catalogue No 69504) as per the protocols provided by the manufacturer. The final templates were kept in -20°C. Similarly, an adult Toxocara canis worm was collected from dog at post mortem and DNA was extracted following the same protocol use for extraction of DNA from D. immitis. The DNA extracted from T. canis was used as negative control. Subsequently, DNA from blood sample, negative for D. immitis based on SNAP®4Dx test (IDEXX Laboratories, Inc., USA), was isolated following the protocol mention above, also stands for negative control.
The DNA samples were subjected to PCR for detection of D. immitis and Wolbachia sp specific genes. D. immitis PCR was performed using external primers to span the internal transcribed spacer region 2 (ITS 2) of the ribosomal DNA by using a forward primer 5’-CATCAGGTGATGATGTGAT-3 and reverse primer 5’-TTGATTGGATTTTAACGTATCATTT-3’ (Rishniw et al., 2006). These primers (D.imm-F1 and D.imm R1) should amplify a 302 bp DNA fragment. The amplification was carried out in 25 µl reaction volume containing 12.5 µl of 2X PCR master mix (Fermentas, USA) containing 0.05 units/µl Taq DNA Polymerase in reaction buffer, 4 mM magnesium chloride, 0.4 mM dATP, 0.4mM dCTP, 0.4 mM dGTP and 0.4 mM dTTP, 0.5 mM primer, 3µl of template DNA(60ng/ µl concentration) and nuclease free water to make 25 µl. The cycling condition used for amplifying the targeted product consisted of a initial denaturing step at 94 0 C for 2 min and 32 cycles of denaturation (30 s at 94 0 C), annealing (30 s at 58 0 C) and extension (30 s at 72 0 C); a final extension (7 min at 72 0 C) and soak at 4°C in a thermal cycler (Applied Biosystem). Again the DNA sample isolated from adult D. immitis is subjected to amplification of FtsZ gene of Wolbachia conserved region by using a forward primer 5’-ATAACAGCAGGAATGGGTGGT-3’ and reverse primer 5’-TCACGCACTCTATTTGCTGCA-3’ (Rossi et al., 2010). The reaction was carried out with same volume and with same quantity of reagents. The reactions were performed with an initial denaturation temperature of 940C for 2 min followed by 40 cycles of denaturation at 940C for 30 s , annealing at 600 C for 1 min, extension at 720 C for 30 s ; and a final extension was then done at 720 C for 10 min. These primers should amplify a 550bp DNA fragment. In both the occasions separate PCR reactions were also run using template DNA from Toxocara canis and DNA from blood sample, negative for D. immitis. The amplified products were then run on 1.2 per cent agarose gel premixed with ethidium bromide (10µg/ml) in submarine gel electrophoresis and visualized under UV-transilluminator. The PCR product amplified at a desired size is then excised and purified from agarose gel by using gel extraction kit (QIAGEN GmbH, Germany). The purified FtsZ DNA of Wolbachia was sent for custom sequencing to M/S Xcelris Labs Ltd., Ahmedabad, India. The identity of the sequence obtained was confirmed by Basic Local Alignment Search Tool (BLAST), NCBI (National Center for Biotechnology Information, USA). Analysis of the sequence was done by using Clustal X2 and Megalign (DNASTAR). The sequences were submitted to public domain in GenBank (NCBI) and phylogenetic relationship study was carried out by using MEGA6 software.
Results and Discussion
In the present work, the evidence of Wolbachia in D. immitis (canine heartworm) was confirmed and genetic characterization of Wolbachia was carried out from the parasite collected from two cities of Northeast India, namely, Guwahati and Aizawl. Confirmation of the adult parasite collected from heart as D. immitis was based on morphological description (Soulsby, 1982) followed by amplification of ITS-2 fragment of ribosomal DNA. As expected, PCR amplification of D. immitis genomic DNA with ITS-2 specific primer yielded distinctive bands at 302bp confirming the quality of template DNA and authenticity of the experimental protocol. Simultaneously, when genomic DNA extracted from blood of uninfected dogs used for PCR, it failed to show any amplification (Fig. 1a).
Amplification of Wolbachia partial FstZ gene was done in order to detect the Wolbachia in adult D. immitis. On PCR amplification, the desire discrete band at 550 bp was visualized both worm DNA as well as in DNA of microfilaremic blood samples on 1.2 per cent agarose gel (Fig. 1b).
Fig.1b: PCR amplification of Wolbachia of D. immitis (wDiFtsZ) (550bp) Lane A: 100bp molecular marker, Lane B, C, D & E: DNA of D. immitis worm Lane F: Negative control, Lane G, H & I: DNA from heartworm positive blood
To confirm the specificity of the test, when template DNA from D. immitis Ag negative dog’s blood samples were run, the PCR reactions were consistently showed absence of any band. This PCR technique hence proved the endosymbiotic relationship of Wolbachia with D. immitis. The finding of the present study is corroborated with the findings of earlier workers (Rossi et al., 2010). The custom DNA sequence was then BLAST to see homologous sequence in the data base. It has been seen that the sequence generated is homologous to the available Wolbachia sequence in the GenBank with 99-100 per cent identity. FtsZ gene of Wolbachia was submitted to GenBank with accession number JX524742. Alignment of the FtsZ gene sequence generated was done by Clustal X2 software and phylogenetic relationship study was done by using MEGA6 software (Fig. 2).
The percent identity of Wolbachia endosymbiont of Guwahati isolate range from 99.4 (AJ131709, AJ010272 and AJ495000) to 99.8 per cent (AY523519). It was observed that Guwahati isolate of Wolbachia has high identity with USA isolate (AY523519) (Fig. 3). On contrary, the percent identity were lesser i.e. 89-89.9 per cent with Wolbachia of Onchocerca, while there is 87.4 per cent identity with Wolbachia of Drosophilla tritis. Based on the present observation, it is hypothesized that the endosymbiont co-evolved along with the host/parasite and play a vital role in the homeostasis of the parasite and therefore the pathogenesis of heartworm.
The endosymbiotic relationship between Wolbachia and Dirofilaria immitis have been established and reported from the North East India.
The authors acknowledge to the Dean, College of Veterinary Sciences &AH, Central Agricultural University, Selesih, Aizawl, Mizoram for providing necessary facilities to carry out the work.