Emerging bacterial infectious diseases/pathogens vectored by human lice.

Human lice have always been a major public health concern due to their vector capacity for louse-borne infectious diseases, like trench fever, louse-borne relapsing fever, and epidemic fever, which are caused by Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii, respectively. Those diseases are currently re-emerging in the regions of poor hygiene, social poverty, or wars with life-threatening consequences. These louse-borne diseases have also caused outbreaks among populations in jails and refugee camps. In addition, antibodies and DNAs to those pathogens have been steadily detected in homeless populations. Importantly, more bacterial pathogens have been detected in human lice, and some have been transmitted by human lice in laboratories. Here, we provide a comprehensive review and update on louse-borne infectious diseases/bacterial pathogens.

The fragmented mitochondrial genomes of two Linognathus lice reveal active minichromosomal recombination and recombination hotspots.

Evidence for recombination between mitochondrial (mt) minichromosomes has been reported in sucking lice, but it is still not clear how frequent mt minichromosomal recombination occurs. We sequenced the mt genomes of the cattle louse Linognathus vituli and the goat louse L. africanus. Both Linognathus species have 10 mt minichromosomes, and seven of them have the same gene content and gene arrangement. Comparison of mt karyotypes revealed numerous inter-minichromosomal recombination events in the evolution of Linognathus species. Minichromosome merger, gene duplication and gene translocation occurred in the lineage leading to Linognathus lice. After the divergence of L. vituli and L. africanus, duplication, degeneration, deletion and translocation of genes also occurred independently in each species. Most of the recombination events in the Linognathus species occurred upstream of either cox3 or nad2, indicating these two locations were hotspots for inter-minichromosomal recombination. Our results provide an important perspective on mt genome evolution in metazoans.

Characterization of the complete mitochondrial genomes of five hard ticks and phylogenetic implications.

Ticks are blood-sucking ectoparasites with significant medical and veterinary importance, capable of transmitting bacteria, protozoa, fungi, and viruses that cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of five hard tick species and analyzed features of their gene contents and genome organizations. The complete mt genomes of Haemaphysalis verticalis, H. flava, H. longicornis, Rhipicephalus sanguineus and Hyalomma asiaticum were 14855 bp, 14689 bp, 14693 bp, 14715 bp and 14722 bp in size, respectively. Their gene contents and arrangements are the same as those of most species of metastriate Ixodida, but distinct from species of genus Ixodes. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes with two different computational algorithms (Bayesian inference and maximum likelihood) revealed the monophylies of the genera Rhipicephalus, Ixodes and Amblyomma, however, rejected the monophyly of the genus Haemaphysalis. To our knowledge, this is the first report of the complete mt genome of H. verticalis. These datasets provide useful mtDNA markers for further studies of the identification and classification of hard ticks.

Characterization of the fragmented mitochondrial genome of domestic pig louse Haematopinus suis (Insecta: Haematopinidae) from China.

The domestic pig louse Haematopinus suis (Linnaeus, 1758) (Phthiraptera: Anoplura) is a common ectoparasite of domestic pigs, which can act as a vector of various infectious disease agents. Despite its significance, the molecular genetics, biology and systematics of H. suis from China have not been studied in detail. In the present study, the entire mitochondrial (mt) genome of H. suis isolate from China was sequenced and compared with that of H. suis isolate from Australia. We identified 37 mt genes located on nine circular mt minichromosomes, 2.9 kb-4.2 kb in size, each containing 2-8 genes and one large non-coding region (NCR) (1,957 bp-2,226 bp). The number of minichromosomes, gene content, and gene order in H. suis isolates from China and Australia are identical. Total sequence identity across coding regions was 96.3% between H. suis isolates from China and Australia. For the 13 protein-coding genes, sequence differences ranged from 2.8%-6.5% consistent nucleotides with amino acids. Our result is H. suis isolates from China and Australia being the same H. suis species. The present study determined the entire mt genome of H. suis from China, providing additional genetic markers for studying the molecular genetics, biology and systematics of domestic pig louse.

Characterization of the complete mitochondrial genome of Culex vishnui (Diptera: Culicidae), one of the major vectors of Japanese encephalitis virus.

Culex mosquitoes (Diptera: Culicidae) can transmit a variety of arthropod-borne viruses (arboviruses), causing human and animal diseases. Cx. vishnui, Cx. pseudovishnui, and Cx. tritaeniorhynchus are three representative species in Culex vishnui subgroup, which are widely distributed in southeast Asia, and they have been proved as the main vectors transmitting Japanese encephalitis virus (JEV) that could cause human infectious mosquito-borne disease across Asia. However, the epidemiology, biology, and even molecular information of those mosquitos remain poorly understood, and only the mitochondrial genome (mitogenome) of Cx. tritaeniorhynchus has been reported in these species. In the present study, we sequenced and annotated the complete mitogenome sequence of Cx. vishnui which was 15,587 bp in length, comprising 37 genes. Comparisons of nucleotide and amino acid sequences between Cx. vishnui and Cx. tritaeniorhynchus revealed that most genes within Culex vishnui subgroup were conserved, except atp8, nad1, atp6, and nad6, with differences of 0.4 (rrnS) - 15.1% (tRNAs) and 0 (nad4L) - 9.4% (atp8), respectively, interestingly suggesting the genes nad4L and rrnS were the most conserved but atp8 gene was the least. The results based on nucleotide diversity also supported a relatively uniform distribution of the intraspecific differences in Cx. vishnui and Cx. tritaeniorhynchus with only one highly pronounced peak of divergence centered at the control region. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes supported the previous taxonomic classification of the family Culicidae and the monophyly of tribes Aedini, Culicini, Mansoniini, and Sabethini. The present study revealed detailed information on the subgroup Culex vishnui, reanalyzed the relationships within the family Culicidae, provided better markers to identify and distinguish Culex species, and offered more markers for studying the molecular epidemiology, population genetics, and molecular phylogenetics of Cx. vishnui.

Morphological and molecular evidence reveals a new species of chewing louse Pancola ailurus n. sp. (Phthiraptera: Trichodectidae) from the endangered Chinese red panda Ailurus styani.

Lice are six-legged, wingless, insect parasites of mammals and birds, and include two main functional groups: blood-sucking lice and chewing lice. However, it is still not clear whether the Chinese red panda Ailurus styani is infested with the parasitic louse. In the present study, we describe a new genus and a species of chewing louse, Pancola ailurus (Phthiraptera: Trichodectidae) based on morphological and molecular datasets. The morphological features showed that Pancola is closer to Paratrichodectes. The genetic divergence of cox1 and 12S rRNA among the Pancola ailurus n. sp. and other Trichodectidae lice was 29.7 - 34.6% and 38.9 - 43.6%, respectively. Phylogenetic analyses based on the available mitochondrial gene sequences showed that P. ailurus n. sp. is more closely related to Trichodectes canis and Geomydoecus aurei than to Felicola subrostratus and together nested within the family Trichodectidae. This study is the first record of parasitic lice from the endangered Chinese red panda A. styani and highlights the importance of integrating morphological and molecular datasets for the identification and discrimination of new louse species.

Aonchotheca (Nematoda: Capillariidae) is validated as a separated genus from Capillaria by both mitochondrial and nuclear ribosomal DNA.

BACKGROUND: The family Capillariidae is a group of thread-like nematodes of 27 genera and over 300 species that infect a great variety of hosts including humans. Among these, some taxa such as the genus Aonchotheca have remained controversial regarding their systematic status for decades. The aim of the current study was to verify Aonchotheca's systemic status and to further determine whether it is a distinct genus from Capillaria using molecular and phylogenetic analyses. RESULTS: We sequenced the mitochondrial (mt) genome and nuclear small subunit (18S) rRNA gene of Aonchotheca putorii, a representative species of the genus, and investigated its systematic status in Trichinellida using maximum likelihood and Bayesian inference. The differences in amino acid sequences of 13 protein-coding genes were 12.69-67.35% among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillaria with cox1 (12.69%) and atp8 (67.35%) as the most and the least conserved gene, respectively, and the difference of two mt rRNAs was 18.61-34.15%. Phylogenetic analyses of the complete mt genome and 18S rRNAs unequivocally showed that Aonchotheca was a distinct genus from Capillaria. CONCLUSIONS: Large difference exists among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillarias. Aonchotheca putorii is the first species in the genus Aonchotheca for which a complete mitogenome has been sequenced. These data are useful for phylogenetics, systematics and the evolution of Capillariidae.

A Novel Mitochondrial Genome Fragmentation Pattern in the Buffalo Louse Haematopinus tuberculatus (Psocodea: Haematopinidae).

Sucking lice are obligate ectoparasites of mammalian hosts, causing serious public health problems and economic losses worldwide. It is well known that sucking lice have fragmented mitochondrial (mt) genomes, but many remain undetermined. To better understand patterns of mt genome fragmentation in the sucking lice, we sequenced the mt genome of the buffalo louse Haematopinus tuberculatus using next-generation sequencing (NGS). The mt genome of H. tuberculatus has ten circular minichromosomes containing a total of 37 genes. Each minichromosome is 2.9-5.0 kb long and carries one to eight genes plus one large non-coding region. The number of mt minichromosomes of H. tuberculatus (ten) is different from those of congeneric species (horse louse H. asini, domestic pig louse H. suis and wild pig louse H. apri) and other sucking lice. Two events (gene translocation and merger of mt minichromosome) are observed in Haematopinus. Compared to other studies, our phylogeny generated from mt genome datasets showed a different topology, suggesting that inclusion of data other than mt genomes would be required to resolve phylogeny of sucking lice. To our knowledge, this is the first report of a ten mt minichromosomes genome in sucking lice, which opens a new outlook into unexplored mt genome fragmentation patterns in sucking lice.

Comparative Mitochondrial Genomic Analysis Robustly Supported That Cat Tapeworm Hydatigera taeniaeformis (Platyhelminthes: Cestoda) Represents a Species Complex.

Hydatigera taeniaeformis is one of the most common intestinal tapeworms that has a worldwide distribution. In this study, we sequenced the complete mitochondrial (mt) genome of H. taeniaeformis from the leopard cat (designated HTLC) and compared it with those of H. taeniaeformis from the cat in China (designated HTCC) and Germany (designated HTCG). The complete mt genome sequence of HTLC is 13,814 bp in size, which is 167 bp longer than that of HTCC and is 74 bp longer than that of HTCG. Across the entire mt genome (except for the two non-coding regions), the sequence difference was 3.3% between HTLC and HTCC, 12.0% between HTLC and HTCG, and 12.1% between HTCC and HTCG. The difference across both nucleotide and amino acid sequences of the 12 protein-coding genes was 4.1 and 2.3% between the HTLC and HTCC, 13.3 and 10.0% between the HTLC and HTCG, and 13.8 and 10.6% between the HTCC and HTCG, respectively. Phylogenetic analysis based on concatenated amino acid sequences of 12 protein-coding genes showed the separation of H. taeniaeformis from different hosts and geographical regions into two distinct clades. Our analysis showed that the cat tapeworm H. taeniaeformis represents a species complex. The novel mt genomic datasets provide useful markers for further studies of the taxonomy and systematics of cat tapeworm H. taeniaeformis.

Mitochondrial phylogenomics provides insights into the taxonomy and phylogeny of fleas.

BACKGROUND: Fleas (Insecta: Siphonaptera) are obligatory hematophagous ectoparasites of humans and animals and serve as vectors of many disease-causing agents. Despite past and current research efforts on fleas due to their medical and veterinary importance, correct identification and robust phylogenetic analysis of these ectoparasites have often proved challenging. METHODS: We decoded the complete mitochondrial (mt) genome of the human flea Pulex irritans and nearly complete mt genome of the dog flea Ctenocephalides canis, and subsequently used this information to reconstruct the phylogeny of fleas among Endopterygota insects. RESULTS: The complete mt genome of P. irritans was 20,337 bp, whereas the clearly sequenced coding region of the C. canis mt genome was 15,609 bp. Both mt genomes were found to contain 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and two ribosomal RNA genes. The coding region of the C. canis mt genome was only 93.5% identical to that of the cat flea C. felis, unequivocally confirming that they are distinct species. Our phylogenomic analyses of the mt genomes showed a sister relationship between the order Siphonaptera and orders Diptera + Mecoptera + Megaloptera + Neuroptera and positively support the hypothesis that the fleas in the order Siphonaptera are monophyletic. CONCLUSIONS: Our results demonstrate that the mt genomes of P. irritans and C. canis are different. The phylogenetic tree shows that fleas are monophyletic and strongly support an order-level objective. These mt genomes provide novel molecular markers for studying the taxonomy and phylogeny of fleas in the future.

Human pediculosis, a global public health problem.

BACKGROUND: Human pediculosis is caused by hematophagous lice, which are transmitted between individuals via direct and/or indirect contact. Despite the public health importance of louse infestation, information concerning the global burden of pediculosis and the epidemiological landscape of louse-borne diseases is limited. The aim of this review was to summarize the biology, epidemiology, diagnosis, and control of lice infestation in humans. We also discussed the latest advances in molecular taxonomy and molecular genetics of lice. METHODS: We searched five electronic bibliographic databases (PubMed, ScienceDirect, CNKI, VIP Chinese Journal Database, and Wanfang Data) and followed a standard approach for conducting scoping reviews to identify studies on various aspects of human lice. Relevant information reported in the identified studies were collated, categorized, and summarized. RESULTS: A total of 282 studies were eligible for the final review. Human pediculosis remains a public health issue affecting millions of people worldwide. Emerging evidence suggests that head lice and body lice should be considered conspecific, with different genotypes and ecotypes. Phylogenetic analysis based on mitochondrial (mt) cytb gene sequences identified six distinct clades of lice worldwide. In addition to the direct effect on human health, lice can serve as vectors of disease-causing pathogens. The use of insecticides plays a crucial role in the treatment and prevention of louse infestation. Genome sequencing has advanced our knowledge of the genetic structure and evolutionary biology of human lice. CONCLUSIONS: Human pediculosis is a public health problem affecting millions of people worldwide, particularly in developing countries. More progress can be made if emphasis is placed on the use of emerging omics technologies to elucidate the mechanisms that underpin the physiological, ecological, and evolutionary aspects of lice.

Comparative analyses of the mitochondrial genomes of the cattle tick Rhipicephalus microplus clades A and B from China.

The cattle tick (Rhipicephalus microplus) is one of the most common ticks parasitizing livestock, causing diseases as the vector of pathogens. In this study, we amplified and sequenced the complete mitochondrial (mt) genome of R. microplus from Hainan province of China and compared it with that of R. microplus from Guizhou province of China. The mt genome sequence of R. microplus from Hainan isolate was 15,163 bp in size, which was significantly longer (299 bp) than R. microplus from Guizhou isolate. Nucleotide sequence difference in the entire mt genome except for non-coding region was 5.6% between R. microplus from Hainan and Guizhou isolates. For the 13 protein-coding genes, this comparison revealed the sequence differences of nucleotide (3.8-10.1%) and amino acid (1.2-17.3%). Phylogenetic analysis of R. microplus indicated that R. microplus from Hainan isolate clustered in clade A, and R. microplus from Guizhou isolate clustered in clade B. Taken together, the findings support the recent proposal the existence of two lineages (clades A and B) of R. microplus in China.

Comparative analyses of the fragmented mitochondrial genomes of wild pig louse Haematopinus apri from China and Japan.

The wild pig louse Haematopinus apri is one of the commonest ectoparasites of wild pigs. In the present study, the entire mitochondrial (mt) genome of wild pig louse H. apri from China was sequenced and compared with previously characterized wild pig louse H. apri from Japan. We identified all of the 37 mt genes in the wild pig louse H. apri from China which are on nine circular minichromosomes. Each mt minichromosome is 2.9 kb-4.2 kb size and contains 2-8 genes and one non-coding region (1543 bp-2534 bp). The number of minichromosomes, gene content and gene order in the both mt genomes of wild pig louse H. apri from China and Japan are the same. The identity of the both mt genomes (except for non-coding regions) was 98.3% between wild pig louse H. apri from China and Japan. The entire mt genome sequence (except for non-coding regions) of wild pig louse H. apri from China is longer (3 bp) than that from Japan. For the 13 protein-coding genes, this comparison showed sequence differences in each gene at both the nucleotide (0.8%-2.4%) and amino acid (0.4%-3.5%) levels. The most conserved of these genes was the nad6, whereas the nad2 was least conserved at the nucleotide levels. This is the first comprehensive comparison of the mt genomes of a louse species from different geographic locations. This useful data provides additional genetic markers to study the phylogeny, systematics and population genetics of wild pig louse H. apri.

Characterization of the complete mitochondrial genomes of six horseflies (Diptera: Tabanidae).

The family Tabanidae (Insecta: Diptera) is one of the economically most important group of haematophagous insects, causing millions of livestock deaths per year. However, current knowledge on the mitochondrial genomes (mitogenomes) from this family is limited. Additional tabanid mitogenomes characterization is of utmost importance for their identification, epidemiologic and phylogenetic studies. We sequenced the mt genomes of six horseflies with an Illumina platform and their phylogenetic relationship was conducted with other infraorder Tabanomorpha members with available mt genome datasets. All six newly sequenced mitogenomes were typical 37-gene circular structures retaining the gene order of Tabanomorpha. The trnQ, trnM and trnA were highly conserved among the six mitogenomes (identity = 100%). The TΨC arm and variable loop regions were relatively more variable compared to the amino acid receptor arm, anticodon arm and DHU arm of the tRNAs. Among 13 protein-coding genes (PCGs) of tabanids mitogenomes, the highest nucleotide diversity was detected in atp8, cox1, cox3, nad6 and cytb (0.1 for each). In addition, atp8 genes exhibited the highest evolutionary rate (ω = 0.24) among 13 PCGs. The interspecies K2P genetic distances among some Tabanus spp. across the mitogenome was greater (0.08) than intergeneric genetic distance between T. amaenus and Atylotus miser (0.07). Phylogenetic analyses revealed non-monophyletic relationships among horseflies of the genus Tabanus. The present study showed mt gene order is highly conserved within Tabanus species. Our mito-phylogenomic analysis supports the paraphyly of the genus Tabanus. The new data provide novel genetic markers for studies of population genetics and systematics of horseflies.

Characterization of the complete mitochondrial genome of the swine kidney worm Stephanurus dentatus (Nematoda: Syngamidae) and phylogenetic implications.

Swine stephanuriasis caused by kidney worm Stephanurus dentatus is a parasitic disease in tropical and subtropical countries, leading to economic losses. Despite its significance as a pathogen, the phylogenetic position and taxonomic status of this nematode remain poorly understood. Mitochondrial (mt) genome sequences are known to provide useful genetic markers for investigations in these areas, but mt genome sequences are lacking for S. dentatus. In the present study, we determined the complete mt genome sequences of S. dentatus with an Illumina platform and compared it with the mt genomes of other closely related species. The circular mt genome was 13,735 bp in size with 36 genes. All genes are transcribed in the same direction and the mt gene arrangement is identified as a GA3 pattern, that is the most common pattern of gene arrangement observed in nematodes to date. Phylogenetic analysis using concatenated amino acid sequences of 12 protein-coding genes supported the hypothesis that S. dentatus was closely related to the family Chabertiidae. Our results provided insights into the phylogenetic relationship of the family Syngamidae within the superfamily Strongyloidea.

The complete mitochondrial genome of capillariid nematodes (Eucoleus annulatus): A novel gene arrangement and phylogenetic implications.

Capillariid nematode is a group of endoparasites of vertebrates with a complex taxonomy, causing significant economic losses to poultry industry. The taxonomic status of the genus Eucoleus remained controversial for several decades. Mitochondrial (mt) DNA provides useful genetic markers for accurate identification of species, but complete mt genome sequences have been lacking for any Capillariid nematodes. In the present study, we decoded the complete mt genome of E. annulatus and examined its phylogenetic relationship with selected members of the class Enoplea nematodes. The circular mt genome of E. annulatus was 14,118 bp, encoded 37 genes with a single non-coding region and showed substantial gene rearrangements (especially tRNA genes) compared to other nematodes studied to date. The complete mt genome of E. annulatus showed a clear A + T bias in nucleotide composition. The number of A (5404) was approximately equal to T (5405) and the GC-skew was negative on average (-0.073). Phylogenetic analyses based on 18S rDNA placed Eucoleus spp. well apart from each other and supported the proposal that Eucoleus and Capillaria are two distinct genera. Similarly, Bayesian inference (BI) and Maximum likelihood (ML) phylogenies based on mtDNA sequences revealed that the family Capillariidae is more closely related to the family Trichuridae than to the family Trichinellidae. This is the first report of the complete mt genome of capillariid nematodes, and it will provide additional genetic markers for studying the molecular epidemiology, population genetics and systematics of capillariid nematodes and should have implications for the molecular diagnosis, prevention, and control of capillariosis in animals.

Highly rearranged mitochondrial genome in Falcolipeurus lice (Phthiraptera: Philopteridae) from endangered eagles.

BACKGROUND: Fragmented mitochondrial (mt) genomes and extensive mt gene rearrangements have been frequently reported from parasitic lice (Insecta: Phthiraptera). However, relatively little is known about the mt genomes from the family Philopteridae, the most species-rich family within the suborder Ischnocera. METHODS: Herein, we use next-generation sequencing to decode the mt genome of Falcolipeurus suturalis and compare it with the mt genome of F. quadripustulatus. Phylogenetic relationships within the family Philopteridae were inferred from the concatenated 13 protein-coding genes of the two Falcolipeurus lice and members of the family Philopteridae using Bayesian inference (BI) and maximum likelihood (ML) methods. RESULTS: The complete mt genome of F. suturalis is a circular, double-stranded DNA molecule 16,659 bp in size that contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and three non-coding regions. The gene order of the F. suturalis mt genome is rearranged relative to that of F. quadripustulatus, and is radically different from both other louse species and the putative ancestral insect. Phylogenetic analyses revealed clear genetic distinctiveness between F. suturalis and F. quadripustulatus (Bayesian posterior probabilities = 1.0 and bootstrapping frequencies = 100), and that the genus Falcolipeurus is sister to the genus Ibidoecus (Bayesian posterior probabilities = 1.0 and bootstrapping frequencies = 100). CONCLUSIONS: These datasets help to better understand gene rearrangements in lice and the phylogenetic position of Falcolipeurus and provide useful genetic markers for systematic studies of bird lice.

Dipylidium caninum draft genome - a new resource for comparative genomic and genetic explorations of flatworms.

Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of ~13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular biology of D. caninum and a new resource for comparative genomic and genetic explorations of this and other flatworms.

The complete mitochondrial genome sequences of the cat flea Ctenocephalides felis felis (Siphonaptera: Pulicidae) support the hypothesis that C. felis isolates from China and USA were the same C. f. felis subspecies.

The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the most important ectoparasite in cats and dogs worldwide. Over the years, there has been much dispute regarding the taxonomic and systematic status of C. felis. Mitochondrial (mt) genome sequences are useful genetic markers for the identification and differentiation of ectoparasites, but the mt genome of C. felis and its subspecies has not yet been entirely characterized. In the present study, the entire mt genome of C. f. felis from China was sequenced and compared with that of C. felis from the USA. Both contain 37 genes and a long non-coding region of >6 kbp. The molecular identity between the Chinese and American isolates was 99%, except for the non-coding region. The protein-coding genes showed differences at both the nucleotide (1.2%) and amino acid (1%) levels. Interestingly, the cox1 gene of the Chinese isolate had an unusual putative start codon (TTT). Taken together, our analyses strongly support the hypothesis that C. felis isolates from China and the USA were the same C. f. felis subspecies. The mt genome sequence of the C. f. felis China isolate presented in this study provides useful molecular markers to further address the taxonomy and systematics of C. felis.