Human sample collection and DNA extraction
Human blood samples positive for Brugia sp. MF by thick night blood smears (NBS) were collected from four districts belonging to the three endemic provinces, (Gampaha and Kalutara from the Western province n=5; Puttalam from the North-western province n=1 and Galle from the Southern province n=1) and from a case detected from a non-endemic district in the North Central province (Anuradhapura). Blood samples positive for Brugia antibodies by the Brugia rapid test were also included in the molecular analysis. The samples were stored at -20° C in EDTA prior to DNA extraction. The MF in blood was concentrated by Nuclepore® membrane filtration and the filter membranes with trapped MF were used for DNA extraction. The DNA was extracted using the ReliaPrep™ Blood DNA Miniprep System (Catalogue number A5082) according to the manufacturer’s instructions [18] with some modifications.
Briefly, a volume of 180µl of PBS was added to polycarbonate membranes with MF and incubated for 45 minutes while shaking (300rpm). A volume of 20μl Proteinase K and 200μl cell lysis buffer solution was then added to the content and incubated at 56°C for 10 minutes and to which 250μl of binding buffer were added. The content was then transferred to ReliaPrep™ binding columns and centrifuged at 5000Xg. The binding columns were transferred to fresh collection tubes and 500μl of column wash solution was added and centrifuged for three minutes at 5000Xg and this was then repeated twice. The binding columns were finally eluted with 50μl Nuclease-Free water. The DNA concentrations was measured by fluorometry according to the instructions (Manual #TM396: www.promega.com/protocols/) and stored at -20°C until used.
DNA amplification
The procedure stated by Rishniw et al., 2006 was used with some modifications using Promega reagents [24]. The pan-filarial primers (DIDR-F1 5'-AGT GCG AAT TGC AGA CGC ATT GAG-3' and DIDR-R1 5'-AGC GGG TAA TCA CGA CTG AGT TGA-3') that spanned the ITS2 of the rDNA designed by Rishniw et al., 2006 was employed to amplify the target DNA region [24]. Known positive and negative controls were included in each PCR reaction. The PCR procedure consisted of an initial denaturing step at 94 °C for 2 min and 32 cycles of denaturing (30s at 94°C), annealing (30s at 58 °C) and extension (30s at 72 °C) and final extension (7 min at 72 °C) in an Eppendorf Mastercycler thermal cycler.
Discrimination of the six species was based on the size of the amplified PCR products. DIDR-F1 and DIDR-R1 primers amplified 484 bp, 542bp, 578 bp, 584 bp, 615 bp and 664 bp products from Dirofilaria repens, Dirofilaria immitis, Acanthocheilonema reconditum, Acanthocheilonema dracunculoides, B. malayi, and B. pahangi respectively [19].
Canine and feline sample processing
The zoonotic surveillance for filarial parasites were done at three locations in the districts of Gampaha (Weliweriya and Wattala) and Puttalam (Madampe) of Western and Northwestern provinces respectively and molecular speciation of the zoonotic Brugia parasite was performed by PCR using panfilarial primers specific for ITS2 region and species confirmed as B. malayi as detailed previously [13].
Mosquito vector analysis
Sample collection, DNA extraction and amplification
The mosquito surveillance was carried in the district of Gampaha in the same areas where zoonotic surveillance was done, using one cattle-baited trap in each location. Mosquitoes of Mansonia sp. were identified using morphological keys. Heads and thoraces of all Mansonia sp. mosquitoes were dissected to identify filarial larvae.
DNA of filarial larvae was extracted using the MightyPrep reagent (Takara Bio Inc, Japan) according to manufacturer’s guide with some modifications as follows: the mosquito heads and thoraces that were positive for filarial larvae were crushed and mounted temporarily on glass slides and flushed with 200μL of MightlyPrep reagent into a microcentrifuge tube. The lysate was homogenized by hard vortexing for 10 seconds. New pipette tips were used each time to prevent cross contamination of samples. The lysates were incubated at 95 °C for 10 minutes. Subsequently the sample lysates were allowed to cool down to room temperature. The cooled lysates were hard-vortexed for another 10 seconds. Finally, the samples were centrifuged at 12,000 rpm for 10 minutes and stored at −20 °C until used for PCR. DNA amplification was done using the same set of primers and procedure used for human and animals’ samples.
DNA sequencing
The Sanger method[20] was applied in cycle sequencing of amplified PCR products (ITS2 region of rDNA) of MF in blood samples of humans (n=2) {Pubudugama (1) and Weliweriya (1)}, canines (n=3) (Pubudugama) and felines (n=6) {Pubudugama (2), Madampe (4)}. Sample selection was based on the concentration of the DNA extracts.
Homology comparison
The DNA sequence homology was analysed using BLASTN. Mega 07 (https://rom yhe studywww.ncbi.nlm.nih.gov/pubmed/27004904) tool and the analysed data was used to generate the phylogenetic tree. Based on NCBI (https://www.ncbi.nlm.nih.gov/) blast, closely related sequences to Brugia sp. genes were retrieved.