Haemaphysalis hoodi (Acari: Ixodidae) on a human from Yaoundé, Cameroon, and its molecular characterization

The genus Haemaphysalis Koch, 1844 (Acari: Ixodidae) is the second-largest genus, with more than 170 described species that primarily parasitize mammals and birds (Guglielmone et al. 2014, Guglielmone et al. 2020). Haemaphysalis species are three-host ticks, mainly distributed in southern and southeastern Asia and tropical Africa (Guglielmone et al. 2014). The present study identified a tick, Haemaphysalis hoodi Warburton & Nuttall, 1909, collected from a human in Yaoundé, Cameroon. This tick species feed on birds in sub-Saharan Africa. To the best of our knowledge, this is the second record of H. hoodi from humans. In addition, 16S ribosomal RNA and cytochrome oxidase I sequences were generated for this species for the first time. Screening pan-Rickettsia-PCR infection gave a negative result.


Introduction
The genus Haemaphysalis Koch, 1844 (Acari: Ixodidae) is the second-largest genus, with more than 170 described species (Guglielmone et al. 2020). Haemaphysalis species are three-host ticks that primarily parasitize mammals and birds (Guglielmone et al. 2014). Species from this genus are mainly distributed in southern and southeastern Asia and tropical Africa, some species are known from Australia, and only a few species occur in the Americas (Guglielmone et al. 2014). Haemaphysalis species are reservoirs and vectors of many pathogenic microorganisms of animals and humans. For instance, Haemaphysalis leachi (Audouin, 1826) transmits Babesia rossi in dogs (Kamani 2021), as well as Rickettsia conorii, which causes human tick-bite fever, and Coxiella burnetii, the causative agent of Q fever (Hoogstraal 1956). Limited studies have been performed on Haemaphysalis species from wildlife that focused on their diversity and role as potential vectors and reservoirs of pathogens.

Material and methods
In late October 2021, a light brown tick was removed manually from the shoulder of a woman in Nkozoa in the Mefou and Afamba Division of the center region of Cameroon (3°52′53.2″N 11°41′54.6″E). The collected tick was transferred to a 1.5 ml tube containing 600 µl absolute ethanol and sent to Bundeswehr Institute of Microbiology, Munich, Germany, for investigation. The tick specimen was first identified using morphological keys (Hoogstraal 1956), under a Keyence VHX-900F microscope (Itasca, IL, USA). DNA was extracted using the QIAamp mini DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. As this is a rare tick species and no sequences from this species are available, 16S rRNA (Halos et al. 2004) and cox I (Apanaskevich et al. 2011) mitochondrial genes were sequenced, and the sequences obtained were edited and compared with the respective sequences deposited in GenBank using BLASTN and phylogenetic analysis. Sequences for each gene were aligned using MAFFT (Katoh and Standley 2013) with default parameters and phylogenetic analyses performed with IQ-Tree2 v1.6.12 (Minh et al. 2020). Optimal evolutionary models were calculated for each gene: 16S (K3Pu + F + I + G4) and Cox1 (TIM2 + F + I + G4). Nodal support was estimated using ultrafast bootstrap (n = 10,000) and the 50% consensus trees were reported. The partial sequences of the mitochondrial 16S rRNA and cox I genes generated in this study for H. hoodi species have been deposited in GenBank under the accession numbers ON189038 and ON191014. Additionally, Rickettsia spp. screening was performed using a previously published real-time PCR assay targeting a part of the gltA gene (Wölfel et al. 2008).

Results and discussion
The tick was identified as a female Haemaphysalis hoodi. The specific characteristics of the female include moderately dense punctations on scutum, broadly salient palpi, and absence of posterodorsal spur on palpal segment II (Fig. 1A,B) (Hoogstraal 1956;Morel 1965). Birds, especially ground feeder birds, are specific hosts for species within the Ornithophysalis subgenus; although some species parasitize birds and mammals, others only parasitize mammals (Hoogstraal 1956;Hoogstraal and Wassef 1973).
Haemaphysalis hoodi is a very rare parasite of humans (Guglielmone and Robbins 2018). Adults of H. hoodi have been found in three cases of human infestation in Ivory Coast although the exact localities were not reported (cited in Guglielmone et al. 2018). Our finding represents the second record of this tick species feeding on humans. Phylogenetic analysis indicated that the 16S rRNA sequence obtained (305 bp) from H. hoodi group in a moderately supported clade with H. bancrofti, H. doenitzi, and H. phasiana (Fig. 2). Of interest is that H. bancrofti is also part of this clade since it is classified in the subgenus Kaiseriana Dias, 1963, while H. hoodi and H. phasiana are classified in the Ornithophysalis Wassef, 1973, subgenus (Hoogstraal andWassef 1973). Similarly, in the cox I analysis (636 bp), H. hoodi group in a well-supported clade with H. bancrofti, H. humerosa, and H. lagostrophi, the latter two species also belonging to the subgenus Ornithophysalis (Hoogstraal and Wassef 1973). While the overall support for the trees was weak, in each tree, several clades with good bootstrap support were obtained. No overwhelming support was found for any of the subgenera as monophyletic lineages. This may be due to limited phylogenetic signal due to the short sequences used in the analysis. It may, however, be noted that a recent analysis using 10 mitochondrial genes also resulted in a paraphyletic Haemaphysalis subgenus (Kelava et al. 2021). As such, more studies that focus on molecular systematics of the Haemaphysalis subgenera are needed to ascertain the validity of various subgenera. Even so, both 16S rRNA and cox I indicate that H. hoodi presents a unique genetic signature compared to other sequences available in the database that shows a genetic relationship to other members of the Ornithophysalis subgenus.
Rickettsia spp. DNA was not amplified in the sample obtained from the H. hoodi female. It would have been expected to detect Rickettsia africae, which is responsible for the African tick-bite fever, mainly transmitted by Amblyomma species or Rickettsia aeschlimannii, a Hyalomma species related ricketsiae. Haemaphysalis species from Africa are not known as vectors for Rickettsia species. Haemaphysalis leachi was supposed to be a vector for Rickettsia conorii in southern Africa, but no isolates are available to confirm this. In Asia, especially in China, many Haemaphysalis Bootstrap support above 80% is indicated and the trees were rooted with Ixodes scapularis. The accession numbers used for the 16S rRNA and cox I genes are indi-cated behind the species names, respectively, and the tick sequenced in the current study is underlined. Subgenera are indicated in parentheses