Morphology,genetic characterization and phylogeny of Aplectana dayaoshanensis n. sp. (Nematoda: Ascaridida) from frogs

Cosmocercoid nematodes are common parasites in the digestive tract of amphibians. However, our knowledge of the species diversity, genetic data and molecular phylogeny of the superfamily Cosmocercoidea are far from being well understood. In the present study, large numbers of cosmocercoid nematodes were collected from the fine-spined frog Sylvirana spinulosa (Smith) (Anura: Ranidae) and the white-spotted thigh tree-frog Polypedates megacephalus (Hallowell) (Anura: Rhacophoridae) in Guangxi Province, China. Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of the genus Aplectana, A. dayaoshanensis n. sp. (Cosmocercoidea: Cosmocercidae). The molecular characterization of small ribosomal DNA (18S), internal transcribed spacer (ITS) and large ribosomal DNA (28S) of A. dayaoshanensis n. sp., together with the 28S of A. chamaeleonis (Baylis, 1929) (collected from Hyperolius kivuensis Ahl in Rwanda), were reported for the first time. Moreover, phylogenetic analyses using maximum likelihood (ML) inference based on 18S + 28S and ITS sequence data, respectively, both supported the family Cosmocercidae to be a monophyletic group and the family Kathlaniidae to be a paraphyletic group. Our phylogenetic results rejected the monophyly of the genus Aplectana. The present results contribute to the knowledge of the species diversity and genetic data of cosmocercoid nematodes, and preliminarily revealed the phylogenetic relationships of the major families and some genera in the Cosmocercoidea.     

Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks.     

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.     

Molecular phylogeny of Amblyomma exornatum and Amblyomma transversale,with reinstatement of the genus Africaniella (Acari: Ixodidae) for the latter

The phylogeny of the hard tick genus Amblyomma Koch, 1844 deserves special attention, because several poorly studied tick species associated with reptiles still bear the name of this genus, although they may not belong to it. This study focuses on the phylogeny of two such species with uncertain taxonomic status, i.e., Amblyomma transversale (Lucas, 1845) and Amblyomma exornatum Koch, 1844, analyzing two mitochondrial (cytochrome c oxidase subunit 1 and 16S rRNA) and two nuclear (18S and 28S rRNA) genes.In the cox1 phylogenetic analysis, both Am. transversale and Am. exornatum were part of a sister group to all other Metastriata, whereas in the 16S rRNA gene analysis, Am. transversale belonged to a sister group to three subfamilies (Amblyomminae Neumann, 1911; Haemaphysalinae Hoogstraal and Aeschlimann, 1982; Bothriocrotoninae Klompen, Dobson and Baker, 2002), and Am. exornatum formed a sister group to other Amblyomminae. However, based on the 18S and 28S rRNA genes, Am. transversale belonged to a sister group of either Bothriocrotoninae alone or of both Bothriocrotoninae and Haemaphysalinae, respectively. In the latter two phylogenetic analyses Am. exornatum always clustered within Amblyomminae. Morphological comparisons revealed that Am. transversale has at least four unique characters and shares a high number of traits with the genera Robertsicus Barker and Burger, 2018 and Archaeocroton Barker and Burger, 2018, as well as with the subgenus Alloceraea Schulze, 1918 (represented by Haemaphysalis inermis Birula, 1895). These results justify that the genus Africaniella Travassos Dias, 1974 should be reinstated, and the species name of Am. transversale should be used as Africaniella transversale (Lucas, 1845).     

Phylogenetic analysis of Babesia species in China based on cytochrome b (COB) gene

In this study, a mitochondrial marker consisting of an approximately 550-bp region of the Cytochrome b genes (COB) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China was 1.6–30.8%. The constructed phylogenetic tree based on the three unlinked gene sequences (partial COB gene, 18S rDNA and ITS) that evolve at different rates by the method of Neighbor-joining revealed the phylogenetic relationship of Babesia species in China compared with other published corresponding sequences from Babesia species. These data indicate that the 18S rDNA more reliably distinguish the deeper branches among some Babesia species than the partial COB gene and ITS, however, the partial COB gene sequence is better for recognizing close lineages among some Babesia species than the 18S rDNA and ITS sequences. So the combined phylogenetic analysis based on the multiple unlinked loci with different evolving rates can facilitate to establish the more reliable phylogenetic relationship of the Babesia genus. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae)

Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera.     

Integrative taxonomy of Mesocriconema onoense (Tylenchida: Criconematidae) from Vietnam highly suggests the synonymization of Mesocriconema brevistylus and related species

The genus Mesocriconema is one of the most diverse genera within the family Criconematidae, known as ring nematodes, with more than 90 species. Although species in this genus usually show distinct morphological characterizations, the identification based only on morphology can lead to misidentification in many studies resulted in a number of synonymizations in the genus over time. In this study, an integrated approach has been applied in characterizing Mesocriconema onoense from Vietnam. The molecular data of 28S rRNA, ITS, 18S rRNA regions were analyzed and discussed to confirm the correct names on GenBank. Besides, phylogenetic analyses of 28S rRNA, ITS, and 18S rRNA regions of Mesocriconema species revealed that Mesocriconema brevistylus should be considered as a junior synonym of M. onoense. Consequently, M. helicus, M. onostris, and M. paronostris should also be considered as the synonyms of M. onoense.     

Evidence for mitochondrial pseudogenes (numts) as a source of contamination in the phylogeny of human whipworms

Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.     

Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain

Background

Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata, and 36 Theileria spp., as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence.

Methods

The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1,400 bp.

Results

A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates, and other species of Theileria available in GenBank. In the constructed tree, Theileria annulata (Iran vaccine strain) was closely related to other T. annulata from Europe, Asia, as well as T. lestoquardi, T. parva and T. taurotragi all in one clade.

Conclusion

Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical), but the similarity of Iran vaccine strain with other T. annulata reported from China, Spain and Italy determined the 97.9 to 99.9% identity.     

Multiplex PCR assay and phylogenetic analysis of sequences derived from D2 domain of 28S rDNA distinguished members of the Anopheles culicifacies complex into two groups,A/D and B/C/E

A multiplex PCR assay was developed using the sequences of the D2 region of 28S ribosomal DNA (rDNA) to discriminate the five members of the Anopheles culicifacies complex provisionally designated as species A, B, C, D and E. Two minus strand primers derived from sequence differences in the D2 variable region and a universal plus strand primer derived from the conserved 28S (rDNA) has delimited five members into species A and D (group 1) and species B, C and E (group 2) in a PCR diagnostic assay. The complete 28S rDNA-D2 region sequence of A. culicifacies sibling species is reported for the first time. Inter-specific sequence divergence was greater than the intra-specific divergence. The phylogenetic relationships inferred from maximum likelihood, maximum parsimony and the neighbor joining analysis confirmed the presence of two unambiguous monophyly clades one consisting of species A and D and the other of species B, C and E and that the A. culicifacies sibling species diverged relatively recently in evolutionary terms despite their considerable differences in bionomics.     

A first insight into the barcodes for African diplostomids (Digenea: Diplostomidae): Brain parasites in Clarias gariepinus (Siluriformes: Clariidae)

Diplostomid trematodes comprise a large and diverse group of widespread digeneans whose larval stages are important parasitic pathogens that may exert serious impacts in wild and cultured freshwater fish. However, our understanding of their diversity remains incomplete especially in the tropics. Our study is the first application of a DNA-based approach to diplostomid diversity in the African continent by generating a database linking sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) barcode region and ITS1-5.8S-ITS2 rRNA gene cluster for brain-infecting diplostomid metacercariae from the catfish Clarias gariepinus. Analyses of newly-generated partial cox1 sequences for 34 larval isolates of Tylodelphys spp. from Tanzania and Diplostomum spp. from Tanzania and Nigeria revealed three strongly supported reciprocally monophyletic lineages of Tylodelphys spp. and one of an unknown species of Diplostomum. The average intraspecific divergence for the cox1 sequences for each recognised novel lineage was distinctly lower compared with interspecific divergence (0.46–0.75% vs 11.7–14.8%). The phylogenetic hypotheses estimated from Bayesian inference and maximum likelihood analyses of ITS1-5.8S-ITS2 data exhibited congruent strong support for the cox1-derived lineages. Our study thus provides molecular-based evidence for the existence of three distinct brain-infecting species co-occurring in natural populations of C. gariepinus. Based on phylogenetic analyses, we re-allocated Diplostomum mashonense Beverley-Burton (1963) to the genus Tylodelphys as a new combination. We also generated cox1 and ITS1-5.8S-ITS2 sequences for an unknown species of Diplostomum from another African fish host, Synodontis nigrita.     

Cytochrome c oxidase subunit III (COX3) gene,an informative marker for phylogenetic analysis and differentiation of Babesia species in China

In this study a 552-bp region of the cytochrome c oxidase subunit III (COX3) was amplified by polymerase chain reaction (PCR) and sequenced from individual Babesia species. Sequence variation between Babesia species from China ranged between 0 and 32.4%. We analyzed the phylogenetic performance of the partial sequence of the COX3 gene to resolve Babesia relationships as compared to the nuclear 18S rRNA and the mitochondrial cytochrome b (COB) gene, These data indicate that the COX3 gene seems to be superior to the COB gene and the 18S rRNA in recognizing close lineages among some Babesia species. Our work indicates that the COX3 gene does complement and corroborate the phylogenetic inferences observed with the nuclear 18S rRNA and the COB gene previously reported. The combined phylogenetic analysis based on the nuclear 18S rRNA and the COX3 gene significantly improved (bootstrap) intraspecies support of the phylogenetic relationship. The presence of additional variable sites in the COX3 gene allowed an improved interspecies differentiation of Babesia species in this study. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.     

Phylogenetic analysis and characterization of a new parasitic cnidarian (Myxosporea: Myxobolidae) parasitizing skin of the giant mottled eel from the Solomon Islands

Myxosporeans are microscopic cnidarians associated with severe diseases in aquaculture and wild fish populations. This group of parasitic cnidarians thus warrants close attention concerning its potential impact on susceptible fish stocks. At present, little is known about this group of parasites infecting anguillid eels. From myxospore specimens collected from a freshwater eel (Anguilla marmorata) in the Solomon Islands, we describe a new species belonging to the genus Myxobolus based on an integrative taxonomic analysis of morphological, biological traits and molecular data. Furthermore, we determined the phylogenetic position and relationships of this species among other platysporine myxosporeans. Molecular phylogenetic assessment of small subunit ribosomal DNA showed that the species clusters together with Myxobolus portucalensis and Echinactinomyxon type 5 Özer, Wootten and Shinn, 2002, in a well-supported subclade. This is the first report of a myxosporean parasite infecting fish from the Solomon Islands.     

Phylogenetic Location of the Spirometra Sparganum Isolates from China,Based on Sequences of 28S rDNA D1

Background

The phylogenetic location of Chinese Spirometra sparganum isolates remains unclear. The aim of this study was to explore the phylogenetic location of the Spirometra sparganum isolates from China.

Methods

The 28S ribosomal DNA (rDNA) D1 sequences of 14 Spirometra sparganum isolates collected from thirteen locations in China were analyzed by using Neighbor-Joining (NJ), maximum parsimony (MP) and Bayesian inference (BI), respectively. To investigate the deep variance of 28S rDNA D1 region among included species, the secondary structure of 28S rDNA D1 region was also calculated using the program RNA structure.

Results

The genus Spirometra as a monophyletic group was evidenced by two inference methods (MP and BI). All sequences within the genus Spirometra had a bulge of a cytosine residue (Bulge C) in the stem 13 of the secondary structure model of 28S rRNA D1 region. Varietal sites in sequences from all thirteen Chinese isolates were appeared in loops. In loops, adenine was the most abundant base (averagely 41.9%) followed by guanine (averagely 30.0%), and cytosine (averagely 15.1%). In stems, the average percentage of G + C (58.3%) was higher than the percentage of A + T (41.7%).

Conclusion

The ‘Bulge C’ in the stem 13 of the 28S rDNA D1 secondary structure could be as a suitable mark to identify the Spirometra species.     

Molecular characterisation of the Microsporidia of the amphipod Gammarus duebeni across its natural range revealed hidden diversity,wide-ranging prevalence and potential for co-evolution

Microsporidia comprise an unusual group of intracellular, eukaryotic parasites that exhibit ubiquitous distribution throughout the animal kingdom. We analysed the small subunit ribosomal gene (SSUrDNA) using PCR and sequencing and screened 894 Gammarus duebeni (Crustacea, Amphipoda) specimens from 35 European marine and freshwater populations. We discovered considerable hidden microsporidian diversity. Blast searches, sequence analysis and phylogenetic analysis revealed intraspecific sequence variants of known species Dictyocoela duebenum, Dictyocoela muelleri, Pleistophora mulleri and Nosema granulosis. For seven SSUrDNA sequences, we did not find corresponding GenBank entries; they likely represent new species, provisionally classified within the genus Microsporidium. Phylogenetic reconstructions revealed their position as polyphyletic, thereby lending support to the hypothesis of an early microsporidian radiation within this host group. Nevertheless, four of the presumptive novel species formed a discrete and well-supported subclade in the phylogenetic analysis. The respective host specimens were collected from disjunct freshwater sites in Wales, Ireland and Brittany (France) and may represent a new, G. duebeni-specific, microsporidian genus. At the population level, our screening showed that parasitism through Microsporidia is the rule rather than the exception in G. duebeni. We found Microsporidia in 91% of sampled G. duebeni populations. This finding may have considerable consequences for the interpretation of results from ecological, behavioural, physiological and evolutionary studies of the host, as parasitism can significantly influence these traits. Because the host G. duebeni has a complex phylogeography and evolutionary history, the studied host–parasite system may have potential as a model system for investigating processes of co-evolution.     

Characterization of the complete mitochondrial genome of Centrorhynchus milvus (Acanthocephala: Polymorphida) and its phylogenetic implications

The phylum Acanthocephala is a small group of obligate parasites of animals. However, the current classifications of Acanthocephala are still under debate. Moreover, our present knowledge of the complete mitochondrial genome of this parasite group remains limited. To fill this knowledge gap, the complete mitochondrial (mt) genome of Centrorhynchus milvus Ward, 1956 (Palaeacanthocephala: Polymorphida) was firstly sequenced and determined based on specimens collected from the red kite (Milvus milvus) in Pakistan. The mitochondrial genome of C. milvus is 14,314 bp in length and contains 36 genes, including 12 protein-coding (PCGs) genes, 22 transfer RNA (tRNA) genes and ribosomal RNA (rRNA) genes (rrnL and rrnS). To elucidate the phylogenetic relationships of the four classes of Acanthocephala and the systematic position of C. milvus, phylogenetic analysis based on concatenated amino acid sequences of 12 PCGs was performed using Bayesian inference (BI). The results supported the monophyly of Archiacanthocephala and Palaeacanthocephala with strong support (BPP = 1) and also indicated that Archiacanthocephala is the sister clade to the remaining classes of Acanthocephala (Palaeacanthocephala, Eoacanthocephala and Polyacanthocephala). However, Polyacanthocephala with only one representative species (P. caballeroi) is nested within Eoacanthocephala. Our phylogenetic analysis also confirmed C. milvus as the member of the family Centrorhynchidae with a sister relationship to C. aluconis. Our present mt genomic data are very useful for studying the molecular epidemiology, population genetics and systematics of acanthocephalans.     

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.     

Whether Dirofilaria repens parasites from South India belong to zoonotic Candidatus Dirofilaria hongkongensis (Dirofilaria sp. hongkongensis)?

The canine and zoonotic dirofilarioses are arthropod-borne parasitic infections caused by nematodes of the genus Dirofilaria, infecting canines, felines and humans throughout the world. Dirofilaria repens was considered as the most common cause of human dirofilariosis in Kerala. In the present study, molecular characterization of Dirofilaria isolates causing dirofilariosis in humans, dogs and jackal from Kerala, South India was undertaken by performing sequence and phylogenetic analysis based on cytochrome oxidase subunit I (COI) gene. The live worms from swellings/ nodules in subconjunctiva or subcutaneous tissue or scrotum were recovered from humans (n = 3), dogs (n = 4) and one jackal. The PCRs targeting a repetitive fragment, 18S rRNA and COI genes yielded products of ~246 bp, ~875 bp and ~350 bp respectively in all the samples. The sequence analysis of 18S rRNA gene revealed the closest identity (98 to 99%) with an already published sequence of D. repens isolated from a human in Japan. However, based on the sequence and phylogenetic analysis of partial sequences of COI gene, the Dirofilaria infecting both animals (dogs, jackal) and humans native to Kerala, South India were identified as genetically conserved and closely related to Dirofilaria sp. hongkongensis. Hence, the results of the present study suggested the existence of Candidatus Dirofilaria hongkongensis (Dirofilaria sp. hongkongensis) in Kerala, South India causing zoonotic filariosis in canines and humans.