The Thai strain of W. bancrofti microfilariae was collected from microfilaraemic Thai patients living in Tha Song Yang District, Tak Province, and Sankhla Buri District, Kanchanaburi Province, in the western region of Thailand. The Myanmar strain of W. bancrofti microfilariae were collected from microfilaraemic Myanmar migrants living in Mae Sot District, Tak Province, in the western region of Thailand. Among 6 Myanmar patients recruited for this study, 3 were from Rangon and 3 from Moulmein, Myanmar (Figure 1).

This study was reviewed and approved by the Ethics Committee of the Faculty of Medicine, Chulalongkorn University (Bangkok, Thailand). As almost none of the Myanmar workers spoke or read either Thai or English, verbal informed consent in the Myanmar language was obtained from each volunteer in the presence of two witnesses. We had a Thai-Myanmar interpreter for communicating with the Myanmar migrants. After the study was completed, all volunteers received a standard course of treatment with diethylcarbamacine.

The periodicity of each strain of W. bancrofti microfilariae was determined by counting the microfilariae from 20 μl finger-prick blood films, in triplicates, taken at 2-hour intervals over a period of 24 hours. We obtained microfilariae from 4 Thai and 4 Myanmar infected cases. Each blood film was stained with Giemsa (Merck, Darmstadt, Germany), as previously described 61819. The blood film was examined under a light microscope by two independent individuals. The average counts of microfilariae were plotted against time.

Blood for morphological and morphometric studies and RAPD was obtained from each infected individual. Ten milliliters of venous blood were collected under sterile technique and universal precautions from Thai patients at 20.00 hours, and from Myanmar patients at 02.00 hours, as previously described 1819.

The standard smear method 20 was used to prepare the slides. Twenty microliters of blood were smeared in a straight line on a clean slide, dried, dehemoglobinized, and stained with Giemsa. The stained microfilariae were assessed by counting the number of nuclei between cephalic space and nerve ring using a light microscope. The body length and width at various sites were measured using an ocular micrometer, the camera-lucida, and curve meter, determined by two independent individuals. Thirty microfilariae from each patient were studied. Differences in means were analyzed by standard t-test using SPSS software program.

One milliliter of blood was mixed with 9 ml of isotonic phosphate buffered saline (PBS [pH 7.4]; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4), filtered through a 5.0 μM Millipore membrane filter (Millipore, Billerica, MA), followed by adding approximately 20 ml of PBS, and air drying 20. After the filters were transferred to the plate containing PBS, microfilariae were harvested using a needle under a stereo microscope, and suspended in 10 μl of PBS.

DNA extraction was performed using FTA paper (Whatman Bioscience, Cambridge, UK) as previously described 21. Briefly, the microfilariae were washed twice with PBS, and then subjected to 3 freeze-thaw cycles, and blotted onto the FTA paper. After air drying, the FTA paper was washed twice with 200 μl of FTA purification buffer (Life Technologies, Gaithersburg, MD) for 15 min, washed twice with 200 μl of TE^-1 buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA [pH 8.0]) for 5 min, and then dried on a heating block at 56°C for 10 min. The dried FTA paper was used as the DNA template in RAPD-PCR amplification.

Six oligonucleotides from the ready-to-go RAPD analysis kit (Amersham Biosciences Ltd., Piscataway, NJ) were used for amplification of random DNA markers to reveal the genetic diversity among W. bancrofti populations. RAPD-PCR was performed with ready-to-go RAPD analysis Beads as described by the manufacturer (Amersham Biosciences). Approximately 10 ng of genomic DNA from microfilriae was used. The RAPD reaction was performed in a DNA thermal cycler (GeneAmp PCR System 2400, Perkin-Elmer, Norwalk, CT), for 1 cycle at 96°C for 4 min, followed by 40 cycles of 94°C for 1 min, 40°C for 1 min and 72°C for 2 min, respectively. The final amplification cycle included 7 min extension at 72°C. Amplified products were analyzed by 2% agarose gel (USB, Cleveland, OH) electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light. The size of each band was determined by Quantity One^® 1-D Analysis Software (Bio-Rad, Hercules, CA). RAPD analysis of human DNA (control), using the same primer and protocol was also conducted to rule out the possibility of contamination of human DNA in the samples. The amplification of all the DNA samples was repeated three times in order to see the variability, if any, in the amplification patterns. Similar patterns were obtained from all experiments.

RAPD profiles were used to measure genetic similarity among W. bancrofti populations. The presence or absence of bands was coded in binary (0, 1) form in a data matrix. Parasites from one individual patient were treated as a genetic population. Relationships among the individual genetic populations of W. bancrofti were determined by a distance matrix method. The approach involved calculation of the Nei (1973) index of genetic similarity 22. Distance values were subjected to phylogenetic analyses using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) as implemented in the UPGMA program of the PAUP software package, version 4.0b10 23.

The periodicity profiles of the Thai and Myanmar strains of W. bancrofti were determined (Figure 2). The microfilariae were present in peripheral blood of 4 Thai patients at all times during a 24-hour period but with higher numbers during the nighttime than during the daytime. This indicated the nocturnal supperiodicity character of Thai strain of W. bancrofti. The peak count of the Thai strain of W. bancrofti microfilariae in peripheral blood was at 20.00 hours.

The microfilariae were present in peripheral blood of 4 Myanmar patients during 18.00 hours and 10.00 hours, and absent during 12.00 hours and 16.00 hours, indicating the nocturnal periodicity character of the Myanmar strains of W. bancrofti. The peak count of the Myanmar strain of W. bancrofti microfilariae in peripheral blood was at 02.00 hours.

Microfilariae of both the Thai and Myanmar strains of W. bancrofti were sheathed, lying in graceful coils without secondary kinking. Somatic nuclei were discrete, overlapping where crowded but with distinct borders, and countable. The nuclear column stopped before the tip of the tail.

Morphometric measurements and counts of nuclei between the cephalic space and nerve ring of the two strains of W. bancrofti microfilariae were demonstrated (Table 1). Some measurement parameters of the Thai strain of W. bancrofti (body length, 299.20 ± 11.87 μm; cephalic space length, 5.47 ± 0.68 μm; and cephalic space width, 5.91 ± 0.53 μm) were significantly (p < 0.05) larger than those of the Myanmar strain of W. bancrofti (the body length, 286.92 ± 9.42 μm; cephalic space length, 5.20 ± 0.71 μm; and cephalic space width, 5.62 ± 0.57 μm). However, other parameters of the Thai strain of W. bancrofti, including head to nerve ring length, 56.81 ± 5.33 μm; Innenkorper length, 42.62 ± 5.79 μm; body width at nerve ring, 6.32 ± 0.52 μm; and number of nuclei between cephalic space and nerve ring, 89.52 ± 6.37, were not statistically different from those of the Myanmar strain of W. bancrofti (head to nerve ring length, 56.24 ± 3.26 μm; Innenkorper length, 42.67 ± 5.09 μm; body width at nerve ring, 6.30 ± 0.69 μm; and number of nuclei between cephalic space and nerve ring, 88.51 ± 8.29).

Out of the six oligonucleotides assayed, only the RAPD Analysis Primer 1 (5'-dGGTGCGGGAA-3') revealed unambiguous genetic polymorphisms between the Thai and Myanmar strains of W. bancrofti, and was retained for the analysis in this study. The RAPD profiles of W. bancrofti from 12 genetic populations (6 Thai patients and 6 Myanmar patients) were analyzed (Figure 3). The analysis of RAPD profiles indicated the existence of genetic variability between the Thai and Myanmar strains of W. bancrofti. The number of DNA fragments amplified in the RAPD-PCR from each sample ranged from 5–9. The fragment sizes of different bands recorded for all the samples ranged from 300–1400 bp. The 300 bp and 795 bp bands were specific to the Myanmar strain of W. bancrofti because the Thai strain did not show these bands. Both strains shared the 645, 705, 1290, and 1400 bp bands. Amplification of human DNA (non-infected with W. bancrofti) with the same primer, under similar conditions, yielded 7 fragments ranging from 470 to 1200 bp, but none of these fragments was identical to the fragments amplified in the W. bancrofti DNA samples (Figure 3). This observation shows that there was no human DNA contamination in the parasite DNA samples used for this study.

A phylogenetic tree was constructed using PAUP software to characterize W. bancrofti populations through phylogenetic analysis. Twelve genetic populations fell into two major clusters (Figure 4). The upper half of tree consisted of 6 genetic populations from the Thai patients. The lower branch consisted of all 6 genetic populations from the Myanmar patients. This phylogenetic tree demonstrated clearly that a genetic diversity existed between the Thai and Myanmar strains of W. bancrofti.