- Open Access
Molecular survey of the head louse Pediculus humanus capitis in Thailand and its potential role for transmitting Acinetobacter spp.
© Sunantaraporn et al.; licensee BioMed Central. 2015
- Received: 11 December 2014
- Accepted: 16 February 2015
- Published: 26 February 2015
Head louse infestation, which is caused by Pediculus humanus capitis, occurs throughout the world. With the advent of molecular techniques, head lice have been classified into three clades. Recent reports have demonstrated that pathogenic organisms could be found in head lice. Head lice and their pathogenic bacteria in Thailand have never been investigated. In this study, we determined the genetic diversity of head lice collected from various areas of Thailand and demonstrated the presence of Acinetobacter spp. in head lice.
Total DNA was extracted from 275 head louse samples that were collected from several geographic regions of Thailand. PCR was used to amplify the head louse COI gene and for detection of Bartonella spp. and Acinetobacter spp. The amplified PCR amplicons were cloned and sequenced. The DNA sequences were analyzed via the neighbor-joining method using Kimura’s 2-parameter model.
The phylogenetic tree based on the COI gene revealed that head lice in Thailand are clearly classified into two clades (A and C). Bartonella spp. was not detected in all the samples, whereas Acinetobacter spp. was detected in 10 samples (3.62%), which consisted of A. baumannii (1.45%), A. radioresistens (1.45%), and A. schindleri (0.72%). The relationship of Acinetobacter spp. and the head lice clades showed that Acinetobacter spp. was found in clade A and C.
Head lice in Thailand are classified into clade A and B based on the COI gene sequences. Pathogenic Acinetobacter spp. was detected in both clades. The data obtained from the study might assist in the development of effective strategies for head lice control in the future. Detection of pathogenic bacteria in head lice could raise awareness of head lice as a source of nosocomial bacterial infections.
- Pediculus humanus capitis
- Acinetobacter spp.
- COI gene
- gltA gene
- rpoB gene
Head lice are obligatory human hematophagous ectoparasites belonging to the Pediculidae family . Head lice infestations or pediculosis occurs throughout the world and is caused by Pediculus humanus capitis [2-4]. Molecular techniques have been used for insect species identification and were applied for the biological, evolutionary, phylogenic, and ecological studies. Mitochondrial genes such as cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) are typically used for insect species identification studies because of the high inter-species variability and low intra-species variation . Previous studies based on the COI and Cyt b genes demonstrated that body lice (P. humanus corporis) and head lice are separated into three clades, A, B, and C . Head lice could be found in a relatively specific geographic distribution for each clade . Clade A has worldwide distribution , clade B is found in Europe, Australia, North America, and Central America , and clade C is found in Nepal , Ethiopia , and Senegal .
Head louse transmission occurs by means of clothing, such as, hats, jackets, and scarves, as well as the shared use of hairbrushes and combs . Several reports have suggested that head lice or body lice infestations are vectors of human diseases , including epidemic typhus, relapsing fever, and trench fever, via infection with the gram-negative bacteria, Rickettsia prowazekii , Borrelia recurrentis , Bartonella quintana , respectively. Peleg et al.  demonstrated Acinetobacter baumannii infections in body and head lice, and the bacteria could cause nosocomial infections and community acquired infections such as pneumonia, bacteremia, endocarditis, and meningitis.
Body lice, instead of head lice, are commonly claimed to be vectors for louse-borne disease transmission because they are associated with high incidences of diseases and high mortality rates, particularly epidemic relapsing fever and typhus, which could be fatal in up to 40% of patients . A recent report demonstrated the detection of body louse-borne pathogens in head lice . Some studies using molecular detection reported that B. quintana DNA could be found in head lice collected from homeless individuals in San Francisco, CA, USA  and Nepalese slum children . Bouvresse et al.  demonstrated A. baumannii in head lice collected from elementary school children in Paris, and Kempf et al.  showed that A. baumannii could be detected in body and head lice collected from healthy individuals from Ethiopia.
In Thailand, data on the genetic identification of head lice and pathogenic bacteria in head lice have not been evaluated. Here, we report the first study of the genetic variations of head lice collected from several geographic regions of Thailand as well as the potential of lice as vectors for Acinetobacter spp.
Head louse collections
The head lice specimens were removed from the 70% ethanol by being washed three times with phosphate buffer saline (1XPBS), and then an individual head louse of each sample was homogenized in 200 μl of lysis buffer G and 20 μl of proteinase K. The genomic DNA was extracted using a DNA extraction kit, Invisorb® spin tissue mini kit (STRATEC molecular GmbH, Berlin, Germany) following the manufacturer’s instructions. The extracted head lice DNA was eluted in 40 μl of elution buffer, and the concentration was measured using Nano drop 2000c (Thermo-scientific, USA). The genomic DNA was stored for an extended time at −20°C until the next stage of the investigation.
Degenerate oligonucleotide primers were designed based on the COI sequences of the head lice (P. humanus capitis) and Phthirus pubis obtained from the GenBank database (GenBank:EU493419, EU493427, EU493433, EU493435, EU493437, EU493439, EU493441 for P. humanus capitis and EF152554, AY696000 to AY696005 for Ph. pubis) as forward primer 5'-GGTACTGGCTGGACTRTTTATCC-3', and the degenerate reverse primer sequences were 5'-CTAAARACTTTYACTCCCGTTGG-3'. The primers were synthesized by 1st BASE Oligonucleotide (Oligo) Synthesis services company (1st BASE Laboratories, Malaysia). PCR was used to detect Bartonella spp. or Acinetobacter spp. The DNA in the head louse samples were targeted from the gltA gene and rpoB gene for Bartonella spp.  and Acinetobacter spp. , respectively. The PCR reaction was set up in a final volume of 25 μl containing approximately 50 ng/μl of extracted DNA, 10 μM of each primer, 10XTaq buffer, 2.5 mM of dNTPs, 2.5 mM of MgCl2 and 1 unit of Taq DNA polymerase (Fermentas, Pittsburgh, PA); double distilled water was the negative control. The PCR amplification conditions were as follows: initial denaturation at 95°C for 3 minutes; 40 cycles of 95°C for 1 minute, 50, 60, and 62°C for COI, gltA, and rpoB gene, respectively for 1 minute and 72°C for 1 minute; and the final extension at 72°C for 7 minutes. The PCR amplicons were determined via 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualized with Quantity One Quantification Analysis Software version 4.5.2 (Gel DocEQ System; Bio-Rad, Hercules, CA).
DNA cloning and sequencing
The PCR amplicons were ligated into pGEM-T Easy Vector (Promega, Madison, WI) using T4 DNA ligase. The recombinant plasmids were transformed into competent cells (Escherichia coli DH5α strain), and then the recombinant plasmids were screened using the blue-white colonies system. The colonies suspected to contain the insert gene were cultured, and the plasmid DNA was extracted using the Invisorb® Spin Plasmid Mini kit (STRATEC molecular GmbH, Berlin, Germany) according to the manufacturer’s instructions. The purified plasmids were sequenced by 1st Base Laboratories, Malaysia.
Sequence analysis and phylogenetic tree construction
The obtained nucleotide sequences were analyzed by comparison with the nucleotide sequence in the GenBank database using BLASTN (http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi), and all the nucleotide sequences from this study were submitted to the GenBank database. The nucleotide sequences of each region were aligned, and the percentage of the intra-specific variation was calculated using BioEdit Sequence Alignment Editor, Version 7.1.9. A phylogenetic tree was constructed via the neighbor-joining method using Kimura’s 2 -parameter model implemented in MEGA6.06, and the tree was tested using 1000 bootstrap replicates and compared with the reference sequence (clade A, B and C). Pediculus schaeffi accession no. AY695999 was an outgroup.
Sequencing and phylogenetic tree analysis
Detection of Bartonella spp. and Acinetobacter spp.
Head lice in clade A and C and Acinetobacter spp. detection in Thailand
Sample no. (n)
Clade A (n)
Clade C (n)
Acinetobacter species (n)
% infection rate in Thailand
A. baumannii (n = 1)
A. radioresistens (n = 2)
A. baumannii (n = 2)
A. baumannii (n = 1)
A. radioresistens (n = 1)
A. schindleri (n = 1)
A. schindleri (n = 1)
A. radioresistens (n = 1)
Head louse infestation remains a health problem of children in Thailand. Using mitochondrial COI gene sequences, we successfully demonstrated genetic variations in head lice collected from different geographical regions of Thailand. The phylogenetic tree analyses on the head louse sequences classified the lice into 2 clades. In accordance with previous reports, Kittler et al. and Reed et al. revealed that the phylogenetic tree analyses of their studies examined the COI sequence data, which showed 3 clades of human lice collected from Europe, Africa, and Asia. One clade contained head and body lice (clade A), whereas clade B and clade C each contained only head lice [9,10,25]. This study demonstrated that head lice in Thailand belong to clade A and C. Both clades could be found in all regions of the country. The data confirm that clade A has worldwide distribution . Previous studies reported that clade C is found in Nepal , Ethiopia , and Senegal ; this is the first report of clade C found in Thailand.
To determine whether head lice could transmit pathogenic bacteria, the primer sets targeting the rpoB gene and gltA gene of Acinetobacter spp.  and Bartonella spp.  were used to detect bacterial DNA in the head louse samples. Three Acinetobacter species (A. baumannii, A. radioresistens and A. schindleri) were detected in 10 samples; 4 samples were positive for A. baumannii, A. radioresistens, and 2 samples were positive for A. schindleri. Previous reports demonstrated that A. baumannii is the most commonly found species in head and body lice in Ethiopia, Portugal, the Netherlands, and France [23,26]. The DNA of A. baumannii was more frequently isolated from body lice than from head lice . Punpanich et al.  reported that children at the Queen Sirikit National Institute of Child Health (QSNICH), Thailand were found to have a nosocomial infection caused by A. baumannii.
B. quintana is a facultative intracellular bacterium that causes diseases including trench fever, chronic bacteremia, endocarditis, bacillary angiomatosis and chronic lymphadenopathy . The bacterial DNA of B. quintana was detected in head lice collected from homeless individuals from USA  and Nepalese slum children ; however, B. quintana DNA was not detected in this study. Our result is similar to that of a previous report conducted on head lice of elementary school children in Paris; the study was unable to detect B. quintana, whereas it found A. baumannii .
The data obtained from this study might be used to develop effective planning for head louse control. The detection of pathogenic bacteria in head lice is useful for monitoring the possible head louse-borne pathogens in humans.
This report is the first study using molecular techniques to investigate head lice in Thailand and the first to describe Acinetobacter spp. in human head lice collected from school children in Thailand. The techniques could be used for classification and determination of the genetic variations of head lice, and the report provides fundamental data for further epidemiological studies of head lice in Thailand. Detection of pathogenic bacteria in head lice is crucial for monitoring the head louse-borne pathogens transmitted to humans. Future studies that include epidemiological data, more geographical areas, and a larger sample size of head lice are required; future studies might provide insights into the evolution of bacteria and vector hosts as well as into whether head lice play a role in spreading Acinetobacter spp. or other pathogens to human hosts.
This study was supported by a Ratchdapisak Sompoj Grant (RA57/081), Faculty of Medicine, Chulalongkorn University, the Thailand Research Fund and Chulalongkron University (RSA 5780024), the Integrated Innovation Academic Center (IIAC) Chulalongkorn University Centenary Academic Development Project and the National Science and Technology Development Agency (Thailand) for a Research Chair Grant.
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