Head lice, Pediculus humanus capitis, occur in four divergent mitochondrial clades (A, B, C and D), each having particular geographical distributions. Recent studies suggest that head lice, as is the case of body lice, can act as a vector for louse-borne diseases. Therefore, understanding the genetic diversity of lice worldwide is of critical importance to our understanding of the risk of louse-borne diseases. Here, we report the results of the first molecular screening of pygmies’ head lice in the Republic of Congo for seven pathogens and an analysis of lice mitochondrial clades. We developed two duplex clade-specific real-time PCRs and identified three major mitochondrial clades: A, C, and D indicating high diversity among the head lice studied. We identified the presence of a dangerous human pathogen, Borrelia recurrentis, the causative agent of relapsing fever, in ten clade A head lice, which was not reported in the Republic of Congo, and B. theileri in one head louse. The results also show widespread infection among head lice with several species of Acinetobacter. A. junii was the most prevalent, followed by A. ursingii, A. baumannii, A. johnsonii, A. schindleri, A. lwoffii, A. nosocomialis and A. towneri. Our study is the first to show the presence of B. recurrentis in African pygmies’ head lice in the Republic of Congo. This study is also the first to report the presence of DNAs of B. theileri and several species of Acinetobacter in human head lice. Further studies are needed to determine whether the head lice can transmit these pathogenic bacteria from person to another.
The head louse, Pediculus humanus capitis, and the body louse, P. h. humanus, are obligatory hematophagous parasite that thrived exclusively on human blood for thousands of years [1,2]. The two lice are now usually considered members of a single species as opposed to separate species [3,4], each louse lives and multiplies in a specific ecological niche: hair for head lice and clothing for body lice [5,6]. Molecular analysis of mitochondrial genes has permitted the classification of Pediculus humanus into three several clades or haplogroups, referred to as A, B, and C [1,2, 7,8, 9]. Haplogroup A is the most common, and possesses a global distribution, including both head and body lice [1,2, 6,8, 9]. Clade B comprises only head lice, is confined to the New Word, Europe, Australia and was recently reported in North and South Africa [2,6, 10,11]. Clade C includes only head lice and is mainly found in Africa and Asia [2,5, 9,10]. Most recently, a novel clade D, comprising both head and body lice, was described in Democratic Republic of the Congo [6]. Prior research suggested that the known lice clades evolved on different lineages of Homo, similarly to those which are known to have existed 2. 3 to 0. 03 million years ago (MYA) [1,11], and accordingly their geographic distribution may provide information regarding the evolutionary history of the lice as well as their human hosts [1,2, 22]. Clade A lice are most likely to have emerged in Africa and to have evolved on the host linage that led to anatomically modern humans (Homo sapiens), showing the signs of a recent demographic expansion out of Africa about 100,000 years ago, first to Eurasia and subsequently to Europe, Asia, and the New World [1,5, 12]. Haplogroup B diverged from haplogroup A between 0. 7 and 1. 2 MYA and may have evolved on archaic hominids, such as the Homo sapiens neanderthalensis, who spread across Europe and Asia, only becoming associated with modern humans during the period of overlap as the result of a recent host switch [1,5, 12]. Head lice are one of the most prevalent parasitic infestations in contemporary populations, particularly in children. They often cause intense itching and, in some cases, insomnia. As a result, they represent a major economic and social concern worldwide [6,13,14]. Body lice, unlike head lice, are nowadays less prevalent and tend to appear mainly in indigent individuals living in poor sanitary conditions [6,9, 13]. They do, however, present a far more serious threat to public health because they transmit at least three deadly bacterial pathogens that have killed millions of peoples, namely: Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis, responsible for epidemic typhus, trench fever, and relapsing fever, respectively [5,9, 13]. Body lice are also suspected of transmitting the agent of plague, Yersinia pestis and the nosocomial pathogen, Acinetobacter baumannii [6,15,16]. Until recently, it was believed that head lice cannot transmit louse-borne diseases [17]. Recently, however, its status as a vector of pathogens has been brought into question, since, they have been found to carry the DNA of B. quintana, B. recurrentis, A. baumannii, and Y. pestis in natural settings [6,18,19,20,21,22,23]. Furthermore, experimental infections have shown that head lice may also act as a vector of louse-borne diseases [24,25], justifying a detailed understanding of their genetic diversity and distribution worldwide. In Central Africa, studies on head lice, particularly those involving indigenous individuals, have received little prior attention. Of these indigenous populations, the African Pygmies are hunter-gatherers who live scattered in the equatorial forest. They are characterized by having a very short stature [26]. The Eastern and Western Pygmies represent the two principal groups of African Pygmies [26]. The Western group is estimated to include 55,000 individuals living in the Western Congo basin, across the countries of Cameroon, Republic of Congo, Gabon and Central African Republic, and its subgroups are identified by different names, including the Binga, Baka, Biaka and Aka or Atsua [26]. Furthermore, the detection of B. recurrentis in African lice remains limited to only a small number of countries. Currently, this bacterium is endemic in Eastern Africa (Ethiopia, Eritrea, Somalia, and Sudan) with the highest number of cases observed in Ethiopia, where it is the seventh most common cause of hospital admission and the fifth most common cause of death [27,28]. Nevertheless, this borreliae has not been reported in any of the Central African countries cited above. In this work, we aimed to study the genetic diversity of head lice collected from African Pygmies in the Republic of Congo and to look for louse-borne pathogens in these lice.
This study was approved by the Health Ministry of the Republic of Congo (000208/MSP/CAB. 15 du Ministère de la Santé et de la Population, 20 August 2015). All necessary permits were obtained from the individuals involved or their legal representatives in the case of children. All permissions were granted orally, because the participants are illiterate. The representatives of a local Health Center and the village elders accompanied the researchers to ensure that information was correctly translated into local languages and that the villagers were willing to take part in the study. A total of 630 head lice samples were collected from 126 apparently healthy authochthonal individuals (pygmies) in the Republic of Congo (Congo-Brazzaville) in August 2015. The collections were conducted in three different villages: i) Thanry-Ipendja, where 137 lice were isolated from 18 people, ii) Pokola, where 163 lice were isolated from 36 people, and iii) Béné-Gamboma, where 330 lice were isolated from 72 people (Fig 1). All the sampled individuals were thoroughly examined for the presence of both head and body lice. All visible head lice were removed from hair using a fine-tooth comb. Lice were then collected from the clean white tissue with forceps. No body lice were found during the examination. All the lice were preserved in 70% ethanol and transported to our laboratory in Marseille (France). The head lice specimens were removed from the 70% ethanol, washed three times in distilled water, and cut in half. The genomic DNA of each half louse was extracted using a DNA extraction kit, QIAamp Tissue Kit (Qiagen SAS, Courtaboeuf, France) with the EZ1 apparatus following the manufacturer’s protocols. The extracted head lice DNA was assessed for quantity and quality using a Nano Drop spectrophotometer (Thermo Scientific, Wilmington, United Kingdom). The genomic DNA was stored at -20°C under sterile conditions until the next stage of the investigation. The qPCR was performed to screen all lice samples using previously reported primers and probes for Borrelia spp. , Bartonella spp. , Acinetobacter spp. , Rickettsia spp. , Rickettsia prowazekii, Y. pestis, and Anaplasma spp. (Table 1). All qPCRs were performed using a CFX96 Real-Time system (Bio-Rad Laboratories) and the Eurogentec Master Mix Probe PCR kit (Eurogentec). We included the DNA of the target bacteria as positive controls and master mixtures as a negative control for each test. We considered samples to be positive when the cycle’s threshold (Ct) was lower than 35 Ct [38]. To identify the species of bacteria, all positive samples from qPCRs for Acinetobacter spp. and Borrelia spp. were further subjected to standard PCR, targeting a portion of the rpoB gene (zone1) and a portion of the flab gene, respectively, using the primers and all conditions as described previously [33,36]. Successful amplification was confirmed via gel electrophoresis and amplicons were prepared and sequenced using similar methods as described for cytb gene for lice above. For comparison, the head lice DNA sequences obtained in this study were combined with the 30 cytb haplotypes reported by Drali et al. [39]. We then complemented this dataset with newly available sequences in GenBank, then assigned them to haplotypes using DnaSP v5. 10 [40]. Finally, we created a dataset that consisted of 51 haplotypes. These haplotypes span 41 geographic locations (countries) in five continents (S1 Table). In order to investigate the possible relationships between the haplotypes, the median-joining (MJ) network using the method of Bandelt was constructed with the program NETWORK4. 6 (www. fluxus-engineering. com/sharenet. htm) [41]. Phylogenetic analyses and tree reconstruction were performed using MEGA software version 6. 06 [42] with 500 bootstrap replications.
A total of 160 head lice cytb sequences were analyzed in this work yielding 83 variable positions defining 15 different haplotypes, including 11 new ones: five from haplogroup A (35. 7%), four from haplogroup D (28. 5%), and six from haplogroup C (42. 8%) (Table 3). These haplotypes, together with references from all the body and head lice haplogroups were used to construct a maximum-likelihood (ML) tree and a median-joining (MJ) network (Figs 2 and 3). ML and MJ analyses had similar results: all the cytb sequences were divided across the four major supported clades, represented by four connected subnetworks distinct groups as shown in the MJ network (Fig 2) corresponding to the known clades: A, D, B, and C. The 15 haplotypes in our study fell into all of the three haplogroups, A, D, and C. The haplogroup A subnetwork was star-like in structure, with the most prevalent and widespread haplotype being A5 (78% of locations and 45. 4% of the 1,005 analyzed human lice) in the center. 24 (15%) of our cytb sequences have this A5 haplotype and are all from the village of Béné-Gamboma, while a total of 64 (40%) cytb sequences (34 sequences from Thanry-Ipendja and 34 sequences from Pokola villages) have the A17 haplotype, which is the second most common A-haplotype and derived from the A5-haplotype by one mutation step. The remaining five clade A sequences, four from Thanry-Ipendja and five from Pokola, defined three novel haplotypes, named here A57, A58, and A59. These three novel haplotypes derived from A17-haplotype by one mutation step. Haplogroup D, which is genetically close to A, only consists of haplotypes from Ethiopia and the Republic Democratic of Congo (RDC). The 45 (45/160) pygmy head lice sequences within clade D defined four haplotypes, of which three are novel (named here: D71, D72, D73), while the fourth haplotype possessed D65 haplotype from RDC. The clade C, representing the most divergent lineage in which two sub-clades can be defined, here referred to as sub-clade C1, which consists of head lice from Ethiopia, France and the Asian continent, and sub-clade C2, which consists of head lice from Senegal and Mali. These two subclades are separated by 12 mutations steps. Interestingly, all 45 (45/160) pygmy head lice sequences within clade C yielded six novel haplotypes, named here as C74-C79 and are parts of sub-clade C1. In this study, the qPCR investigation of all 630 lice samples for Bartonella spp. , Rickettsia spp. , R. prowazekii, Y. pestis, and Anaplasma spp. produced no positive results. However, we obtained positive results when testing for the presence of Borrelia spp. and Acinetobacter spp. The DNA of Borrelia spp. was detected in 11/630 (1. 74%) head lice collected from 7/126 (5. 55%) individuals. All Borrelia-positive lice were clade A and found only in Pokola. The DNA of Acinetobacter spp. was detected in 235/630 (37. 3%) head lice collected from 93/126 (73. 8%) people. Of the 235 positive lice, 176 (26%) were clade A, 24 (3. 8%) clade D, and 47 (7. 5%) clade C. Sixty-one of these infected lice were from Pokola, forty-one from Thanry-Ipendja, and one hundred and thirty-three from Béné-Gamboma.
Here, we report the first molecular data on human head lice, P. h. capitis, infesting the pygmy population in the Republic of Congo in Western Africa. In this study, we established and evaluated for the first time, qPCR assay based on two duplex designed from the cytb gene, which is very well established in the study of lice, in order to identify all known clades of P. humanus. The assay adopted herein proved itself to be fast, specific, sensitive and fully compatible when routinely analyzing large collections of lice specimens. The mtDNA analysis of 630 head lice, collected from 126 pygmies, showed the presence of three major mitochondrial haplogroups: A, C and D, indicating high mtDNA diversity among the head lice studied. Haplogroup A was the most prevalent (56%) followed by haplogroup C (5%). The data confirm that clade A has worldwide distribution, as reported by others [6,8, 9,10]. Previous studies reported that clade C is limited to Nepal and Thailand [1,5, 23], Ethiopia, Senegal and Mali [5,9, 18,22]; this is the first report of clade C which has been found in the Republic of Congo. The remaining samples (10. 3%) were from new haplogroup D, which is known only to exist in Democratic Republic of the Congo and Ethiopia [2,6]. In addition to inter-haplogroup diversity, P. humanus also presents intra-haplogroup diversity, illustrated by many distinct A, B and C haplotypes [2,12,39]. These results are supported by our finding, that, of the 160 head lice cytb sequences analysed, 15 different haplotypes were identified, of which 11 were novel. B. recurrentis is the known causative agent of relapsing fever which, if untreated, can be fatal in up to 40% of patients [13,43,44]. It has long been established that body lice are the main vector for this bacterial pathogen [13,27]. In the present study, the DNA of B. recurrentis was detected in 10/630 (1. 58%) head lice belonging to clade A collected from 6/126 (4. 76%) individuals. Specifically, all positives cases were only found in Pokola, suggesting that a small, unnoticed outbreak may have occurred in the population in this area. This is the second report of the presence of B. recurrentis DNA in human head lice. Recently, this bacterium was also detected in 23% of head lice clade C from patients with louse-borne relapsing fever in Ethiopia and, because these patients were also infested with body lice, the authors hypothesize that head lice might be contaminated by blood that is infected with B. recurrentis [21]. In this study, the discovery of B. recurrentis in the clade A head lice, the same clade that includes body lice, and the absence of body lice may support the hypothesis that B. recurrentis may be transmitted by clade A head lice. Nevertheless, evidence for the presence of the DNA of this bacterium in head lice by PCR cannot distinguish between transient infections, accidentally acquire the pathogen from the blood of infected individuals, and those established in a competent vector, maintain and transmit the pathogen. Further studies are needed to determine whether the head louse can act as a vector of B. recurrentis. Interestingly, one of the Borrelia-positive lice was identified as B. theileri. This is the first report of the presence of the DNA of this species in human head lice. B. theileri is a spirochete that causes borreliosis in cattle, a relapsing fever-like illness, transmitted by hard ticks, such as Rhipicephalus (Boophilus) [45]. This infection can be considered as being rediscovered, appears to exist in regions where diagnostic ability is limited and its impact on livestock is largely unexplored [45]. In this study, two hypotheses can arise from the detection of B. theileri in human head lice. The first one is that the presence of this bacterium results from environmental and/or laboratory contaminations. This hypothesis is hardly possible, because, our work was carried out in a laboratory where B. theileri had never been worked on, nor had B. theileri DNA been extracted. Indeed, each PCR assay was systematically validated by the presence of positive and negative controls. Moreover, our collection contains lice only and didn’t contain another specimens like ticks that could be an important source of environmental contamination. The second hypothesis is that, as head lice feed only on human blood [5], the acquired infection would be from the blood of patients with ongoing bacteremia. Although, humans infected with this spirochete have not been described in the literature, the transmission of this pathogen to humans may not be ruled out. Moreover, the sequence generated in this study was more similar by flaB sequence comparison to those reported from Ornithodoros sp. soft tick (GenBank KP191621) collected from cave in Israel, than, those reported from Rhipicephalus hard tick (GenBank KF569936) from Mali, as shown in the phylogenetic tree (Fig 4). Ornithodoros ticks can feed from multiple warm-blooded vertebrates, including humans, and are known to transmit several species of Borrelia to humans [27,43], thus taking in consideration that the epidemiology of B. theileri is not yet completely discovered, hypothetically it may be transmitted to humans. Finally, if our hypothesis of B. theileri bacteremia in persons harboring head lice is true, this may merely reflect ‘accidental spill-over’ from animal hosts infection, such phenomena has already been described in the literature, with the finding of the DNA of B. duttonii, the species that is only know to infect ticks and humans, in chickens and swine living close to their human owners [43]. Findings from this study also show widespread infection of head lice with several species of Acinetobacter. In total, eight Acinetobacter species were detected in 144 samples; A. junii was the most prevalent, followed by A. ursingii, A. baumannii, A. johnsonii, A. schindleri, A. lwoffii, A. nosocomialis and A. towneri. The DNA of A. towneri was only found in clade C head lice, the DNA of A. lwoffii was only found in clades A and D, while the DNA of the remaining species was found in all three clades A, D and C. Previous studies demonstrated that A. baumannii is the most commonly found species in body and head lice [23], as shown by its detection in 21% of body lice collected worldwide [15], in 33% of head lice collected from Parisian elementary school children, belonging to the clade A [19] and in 71% body and 47% head lice collected from healthy individuals from Ethiopia [20]. Another study, performed in head lice samples collected from elementary school children in Thailand, showed the presence of the DNA of three Acinetobacter species in 3. 62% head lice belonging to both clade A and C. The Acinetobacter species identified were A. baumannii, A. schindleri and A. radioresistens [23]. When comparing the panel of Acinetobacter species found in all these studies with our findings, A. radioresistens was the only species that we did not identify in our head lice specimens. Conversely, our sampling showed, for the first time, the presence of the DNA of A. junii, A. ursingii, A. johnsonii, A. lwoffii, A. nosocomialis and A. towneri in human head lice, but further study is needed to determine the significance of this finding. Furthermore, it is still unknown how these lice acquire their Acinetobacter infections. Some authors have argued that the infection could occur after the ingestion of infected blood meal from individuals with ongoing bacteremia, or may possibly be derived from superficial contamination through human skin while feeding [15]. An experimental study showed that the human body louse, feeding on bacteremic rabbits, is able to acquire and maintain a persistent life-long infection with A. baumannii and A. lwoffii [46]. Furthermore, another study performed a comparison between two sequenced genomes of A. baumannii and showed that the A. baumannii SDF strain, isolated from a human body louse, had several hundred insertion sequence elements which have played a crucial role in its genome reduction (gene disruptions and simple DNA loss) compared to the human multidrug-resistant A. baumannii AYE strain, and also been shown to have low catabolic capacities, suggesting the specific adaptation of this strain to the louse environment [47]. However, Acinetobacter species are widespread in nature (water, soil, living organisms, and the skin of patients and healthy subjects) [47], and because the frequency of with which these species associate with the skin of pygmy population is unknown, it is not possible to rule out the infection of lice by external contamination. Clinically, A. baumannii is known to be a major cause of nosocomial infections in humans and it is an increasing public health concern due to the increasing resistance to antibiotic treatment which has been identified worldwide [47]. Other Acinetobacter species include A. lwoffii and A. junii are also often identified as the cause of infection in humans [48]. However, it still not clear whether these Acinetobacter strains present in lice are the same as those that are responsible for human infections [20].
In conclusion, the qPCR adopted in this study proved to be a fast, sensitive and specific tool that is fully compatible when routinely analyzing a large collections of lice specimens. Our results showed the presence of three major mitochondrial haplogroups: A, C and D, indicating high mtDNA diversity among the pygmy head lice studied. We identified the presence of a dangerous human pathogen, B. recurrentis, the causative agent of relapsing fever, in ten clade A head lice, which had not previously been reported in the Republic of Congo. Findings from this study also show the widespread infection of head lice with several species of Acinetobacter. Despite several investigations into the transmissibility of numerous infectious agents, no conclusive evidence has demonstrated the transmission of disease by head lice. That said, we believe that pathogens detected in head lice may be an indirect tool for evaluating the risk of louse-borne diseases in humans.
Head lice, Pediculus capitis humanus, and body lice, Pediculus h. humanus, are obligatory ectoparasites that feed exclusively on human blood. Currently, the body louse is the only recognized vector of at least three deadly bacterial pathogens that have killed millions of peoples, namely: Rickettsia prowazekii, Bartonella quintana and Borrelia recurrentis, responsible for epidemic typhus, trench fever and relapsing fever, respectively. In this work, we aimed to study the genetic diversity of head lice collected from African Pygmies in the Republic of Congo and to look for louse-borne pathogens in these lice. We detected B. recurrentis in head lice belonged to clade A that is prevalent in the Republic of Congo. Our study also show, for the first time, the presence of DNAs of B. theileri and several species of Acinetobacter in human head lice. Despite several investigations into the transmissibility of numerous infectious agents, no conclusive evidence has demonstrated the transmission of disease by head lice. That said, we believe that pathogens detected in head lice may be an indirect tool for evaluating the risk of louse-borne diseases in humans.
invertebrates
borrelia infection
medicine and health sciences
pathology and laboratory medicine
evolutionary biology
acinetobacter infections
pathogens
population genetics
microbiology
animals
bacterial diseases
body lice
population biology
insect vectors
bacteria
haplogroups
bacterial pathogens
infectious diseases
medical microbiology
epidemiology
microbial pathogens
borrelia
insects
disease vectors
head lice
arthropoda
acinetobacter
haplotypes
genetics
biology and life sciences
lice
organisms
Head Lice of Pygmies Reveal the Presence of Relapsing Fever Borreliae in the Republic of Congo