Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis.

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

Reassessing Stephanofilaria stilesi dermatitis in cattle, with characterization of molecular markers for confirming diagnosis.

BACKGROUND: Stephanofilaria stilesi is a vector-borne filarioid nematode of cattle in North America that is transmitted via the hematophagous horn fly (Haematobia irritans) intermediate host. Despite being relatively common, little attention has been given to a thorough description of S. stilesi lesions and the potential integration of pathological and molecular diagnostic findings to confirm infection. METHODS: To characterize the cutaneous lesions caused by S. stilesi in cattle (Bos taurus taurus and Bos taurus indicus), skin of the ventral abdominal midline was collected from 22 animals during postmortem examination. Skin samples were processed for histology, transmission electron microscopy (TEM), DNA extraction, PCR, and Sanger sequencing targeting molecular markers cytochrome oxidase c subunit 1 (cox1), 12S, 18S rDNA, and 28S rDNA. RESULTS: Macroscopically, lesions ranged from 5 × 4 cm to 36 × 10 cm, consisting of one large single lesion, or two to four ovoid areas at the ventral abdominal midline, surrounding the umbilicus. Each lesion presented as ulcerative dermatitis with dry, serocellular crusts, or alopecic and lichenified areas. Histologically, eosinophilic, neutrophilic, and ulcerative dermatitis with furunculosis, folliculitis, and epidermal hyperplasia was observed. Cross sections of adult nematodes were identified in ~ 60% of the cases (n = 13) within intact follicles, sebaceous ducts, crusts, and areas of furunculosis. Stephanofilaria first-stage larvae (L1) were observed in five cases within "vitelline membranes" in the superficial dermis and crusts. Ultrastructurally, the L1 cross sections were compounded of smooth multilayered cuticle and somatic cells. The "vitelline membrane" is a tri-layered membrane where L1 are suspended in a matrix. Stephanofilaria stilesi DNA was found in 5 out of the 13 cases in which adults or L1 were histologically observed (38%) and in 1 out of the 9 cases without adults or L1 present (11%). Phylogenetic analyses suggest a closer relationship of the genus Stephanofilaria with Thelazioidea, instead of the family Filariidae (Filarioidea), in which it has been historically allocated. CONCLUSIONS: Our study improved the characterization of lesions and described ultrastructural findings of S. stilesi and highlights that molecular tools should be utilized in combination with histology for improved diagnostic resolution.

Heartworms in Halichoerus grypus: first records of Acanthocheilonema spirocauda (Onchocercidae; Filarioidea) in 2 grey seals from the North Sea.

The assumed definitive host of the heartworm Acanthocheilonema spirocauda (Onchocerdidae; Filarioidea) is the harbour seal (Phoca vitulina). This filaroid nematode parasitizing in cardiac ventricles and blood vessel lumina of harbour seals (P. vitulina) has a low prevalence and seldom causes severe health impacts. The seal louse (Echinophthirius horridus) is the assumed intermediate host for transmission of A. spirocauda filariae between seals, comprising a unique parasite assembly conveyed from the terrestrial ancestors of pinnipeds. Although grey seals (Halichoerus grypus) are infected by seal lice, heartworm infection was not verified. Analysing a longterm dataset compiled over decades (1996­2021) of health monitoring seals along the German coasts comprising post mortem investigations and archived parasites, 2 cases of A. spirocauda infected male grey seals were detected. Tentative morphological identification was confirmed with molecular tools by sequencing a section of mtDNA COI and comparing nucleotide data with available heartworm sequence. This is the first record of heartworm individuals collected from the heart of grey seals at necropsy. It remains puzzling why heartworm infection occur much less frequently in grey than in harbour seals, although both species use the same habitat, share mixed haul-outs and consume similar prey species. If transmission occurs directly via seal louse vectors on haul-outs, increasing seal populations in the North- and Baltic Sea could have density dependent effects on prevalence of heartworm and seal louse infections. It remains to be shown how species-specificity of filarial nematodes as well as immune system traits of grey seals influence infection patterns of A. spirocauda.

Molecular detection of Cercopithifilaria, Cruorifilaria and Dipetalonema-like filarial nematodes in ticks of French Guiana.

Filarial nematodes of the Dipetalonema lineage are widespread parasites and include some species that are transmitted by ticks. In this study, we conducted a large molecular survey of ticks in French Guiana, South America, to understand the overall diversity of tick-borne filarioids in this remote region largely covered by dense tropical forests. Out of 682 ticks belonging to 22 species and 6 genera, 21 ticks (3.1%) of the species Amblyomma cajennense, A. oblongoguttatum, A. romitii, Ixodes luciae and Rhipicephalus sanguineus sensu lato were positive for infection by filarioids. Molecular typing and phylogenetic analysis identified all these filarioids as members of the Dipetalonema lineage. While the filarioid of R. sanguineus sensu lato is a previously described species, the canine worm Cercopithifilaria bainae Almeida & Vicente, 1984, all other filarioids detected in this study are related but distinct to already known species in the genera Cercopithifilaria, Cruorifilaria and Dipetalonema. Their vertebrate host range may include a wide variety of mammals present in French Guiana, but dogs, capybaras, and opossums are the best candidate hosts for some of these filarioids. Although the detection of members of the Dipetalonema lineage in ticks of significant medical or veterinary interest is of concern, the risk of contracting a tick-borne filarial infection is still largely unknown. The pathogenicity of these filarioids, their epidemiology, developmental cycles, and mechanisms of transmission by South American tick species now require further study.

Title: Détection moléculaire des nématodes filaires de type Cercopithifilaria, Cruorifilaria et Dipetalonema chez les tiques de Guyane française. Abstract: Les nématodes filaires de la lignée Dipetalonema sont des parasites répandus dont plusieurs espèces sont transmises par les tiques. Dans cette étude, nous avons mené une vaste surveillance moléculaire des tiques en Guyane française, en Amérique du Sud, afin de caractériser la diversité des filaires transmis par les tiques dans cette région largement couverte de forêts tropicales denses. Sur 682 tiques appartenant à 22 espèces et 6 genres, 21 tiques (3.1 %) des espèces Amblyomma cajennense, A. oblongoguttatum, A. romitii, Ixodes luciae et Rhipicephalus sanguineus sensu lato étaient positives pour la détection des filaires. Le typage moléculaire et l'analyse phylogénétique ont permis d'identifier toutes ces filaires comme des membres de la lignée Dipetalonema. Alors que la filaire de R. sanguineus sensu lato est une espèce décrite, la filaire canine Cercopithifilaria bainae Almeida & Vicente, 1984, toutes les autres filaires détectées ici sont apparentées mais distinctes des espèces déjà connues au sein des genres Cercopithifilaria, Cruorifilaria et Dipetalonema. Leur spectre d'hôtes vertébrés pourrait inclure une grande variété de mammifères présents en Guyane française, mais les chiens, les capibaras et les opossums sont les hôtes candidats probables pour certaines de ces filaires. Bien que la détection de membres de la lignée Dipetalonema chez des tiques d'intérêt médical ou vétérinaire soit préoccupante, le risque de contracter une filariose à tiques est encore largement inconnu. La pathogénicité de ces filaires à tiques, leur épidémiologie, leurs cycles de développement et les mécanismes de transmission par les espèces de tiques sud-américaines doivent maintenant être étudiés plus en détail.

Pathological findings associated with Dipetalonema spp. (Spirurida, Onchocercidae) infection in two species of Neotropical monkeys from Brazil.

Among vector-borne helminths, filarioids of the genus Dipetalonema (Spirurida: Onchocercidae) localize in several tissues and body cavities of several animal species, causing mild to moderate lesions. The pathological findings associated with Dipetalonema spp. infection in Neotropical monkeys from southern Brazil are herein described, along with a fatal case due to filarial polyserositis and entrapment of an intestinal segment. At necropsy, nematodes were observed in abdominal and thoracic cavities, or in the pericardium of 37 (31.3%) out of the 118 individuals examined (i.e., 35 Alouatta guariba clamitans and two Sapajus nigritus). In addition, at histology, 27.0% of positive animals presented microfilarie (inside blood vessels of lung, spleen, liver, and brain) and 8.1% presented adult nematodes in the heart, lung, and liver. In two cases, cross-sections of filarioids were associated with areas of epicardial thickening with intense fibrosis and pyogranulomatous inflammation in the brain, heart, liver, lungs, or spleen. The DNA fragment was amplify using the cox1 gene, sequenced and analyzed to identify the nematode species collected; presence of Wolbachia was assessed in the filarioids using the 16S rRNA gene. At BLAST analysis of the cox1 gene, 10 sequences showed 91.7% nucleotide identity with Dipetalonema gracile, and two with D. gracile (98.5%) and Dipetalonema graciliformis (98.3%). Phylogenetic analyses clustered sequences of the cox1 obtained in this study in two clades corresponding with the host species. Wolbachia sp. endosymbiont was detected in four samples. Data herein reported provide a description of pathological lesions associated with the infection by Dipetalonema spp., suggesting that they may cause disease in Neotropical monkeys. In addition, a better understanding of diversity and biology of Dipetalonema spp. in South America is needed to assess the impact they may cause in native non-human primates from Brazil.

Macrophages show up in style when Th2 lymphocytes organize their homecoming.

Monocytes can differentiate into tissue-resident pleural macrophages, but the mechanisms underlying this process are not yet fully understood. In this issue of Immunity, Finlay et al.1 show that Th2 cytokines promote this differentiation in resistant mice infected with Litomosoides sigmodontis.

Occurrence of Dirofilaria repens in wild carnivores in Poland.

Dirofilaria repens is an expanding vector-borne zoonotic parasite of canines and other carnivores. Sub-clinically infected dogs constitute the most important reservoir of the parasite and the source of infection for its mosquito vectors. However, occurrence of D. repens infection in wild animals may contribute to the transmission of the parasite to humans and may explain the endemicity of filariae in newly invaded regions. The aim of the current study was to determine the occurrence of D. repens in 511 blood and spleen samples from seven species of wild carnivores (wolves, red foxes, Eurasian badgers, raccoons, raccoon dogs, stone martens, and pine martens) from different regions of Poland by means of a PCR protocol targeting the 12S rDNA gene. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodeships in four of the seven regions of Poland: Masovia, Lesser Poland, Pomerania and Warmia-Masuria. The highest prevalence was found in Masovia region (8%), coinciding with the highest previously recorded prevalence in dogs in Central Poland. The DNA of Dirofilaria was detected in 16 samples of three species (total prevalence 3.13%). A low and similar percentage of positive samples (1.9%, 4.2% and 4.8%) was recorded among badgers, red foxes, and wolves, respectively. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodships. Based on detection in different voivodeships, D. repens-positive animals were recorded in four out of the seven regions of Poland: in Masovia, Lesser Poland, Pomerania, and Warmia-Masuria. The highest prevalence of filariae was found in Masovia region (8%), reflecting the highest previously recorded prevalence in dogs (12-50%) in Central Poland. In summary, we conducted the first comprehensive study on the epidemiology of D. repens in seven species of wild hosts in all seven regions of Poland and identified the first case of D. repens infection in Eurasian badgers in Poland and the second in Europe.

Filarial infections compromise influenza vaccination efficacy: Lessons from the mouse.

Helminth parasites infect more than a quarter of the human population and inflict significant changes to the immunological status of their hosts. Several human studies report impaired responses to vaccinations in helminth-infected individuals. Analysing the impact of helminth infections on the efficacy of influenza vaccinations in the mouse system helps to elucidate the underlying immunological processes. Concurrent infection with the parasitic nematode Litomosoides sigmodontis reduced the quantity and quality of antibody responses to vaccination against seasonal influenza in BALB/c and C57BL/6 mice. This led to impaired vaccination-induced protection against challenge infections with the human pathogenic 2009 pandemic H1N1 influenza A virus in helminth-infected mice. Impaired responses were also observed if vaccinations were performed after immune-driven or drug-induced clearance of a previous helminth infection. Mechanistically, the suppression was associated with a systemic and sustained expansion of IL-10-producing CD4+CD49b+LAG-3+ type 1 regulatory T cells and partially abrogated by in vivo blockade of the IL-10 receptor. In summary, these findings raise the concern that individuals in helminth-endemic areas may not always benefit from vaccinations, even in the absence of an acute and diagnosable helminth infection.

T helper 2 cells control monocyte to tissue-resident macrophage differentiation during nematode infection of the pleural cavity.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

Filariasis in dogs brought to the Veterinary Teaching Hospital, University of Peradeniya, Sri Lanka.

Dirofilariasis is the predominant emerging zoonotic filariasis in the world. The two most frequent filarial worms that infect dogs are Dirofilaria repens and Dirofilaria immitis. This study reports filariasis among dogs brought to the Veterinary Teaching Hospital (VTH) at the University of Peradeniya and signifies the first molecular characterization of D. repens, responsible for an emerging zoonotic filarial disease in Sri Lanka. Blood samples were collected and were morphologically analyzed using Modified Knott's Technique, followed by molecular analyses. The difference in filariasis prevalence among gender, breed, and age categories was analyzed using a chi-square test. Infection intensities were analyzed using the Mann-Whitney U test and the Kruskal Wallis test. The dogs were brought to the clinic for either vaccination and/or for a regular checkup, and most were sick having non-specific clinical signs. Among the 87 dogs tested, 27.6% were positive for Dirofilaria. Conventional PCR and bi-directional sequencing of genomic DNA of microscopically tested positive samples revealed that the species in Sri Lanka was D. repens. The infection was significantly higher in males (39.1%) than in females (14.6%; χ2 = 0.447, p = 0.011), though it is not significant between puppies (age < 1 year) and adult dogs. More crossbred dogs were infected compared to older and purebred dogs. There was no difference in intensity of infection based on their gender, age, or breed. Sequences obtained from the current study were unique and were only 63% identical to those of D. repens reported from South India. The high number of Dirofilaria infections in domestic dogs indicates a potential reservoir for emerging human dirofilariasis cases in Sri Lanka. Thus, morphological and molecular diagnosis, along with epidemiological assessment of these zoonoses, is critical for the formulation of effective public health programs and control mechanisms.

Unbalanced Arginine pathway and altered maturation of pleural macrophages in Th2-deficient mice during Litomosoides sigmodontis filarial infection.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα^-/-/Il-5^-/- ) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα^-/-/Il-5^-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα^-/-/Il-5^-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80^intermediate macrophages from infected Il-4rα^-/-/Il-5^-/- mice failed to mature into resident F4/80^high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα^-/-/Il-5^-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

Eosinophils in filarial infections: Inducers of protection or pathology?

Filariae are parasitic roundworms, which can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Lymphatic filariasis, also known as elephantiasis, and onchocerciasis, commonly referred to as river blindness, can lead to stigmatizing pathologies and present a socio-economic burden for affected people and their endemic countries. Filariae typically induce a type 2 immune response, which is characterized by cytokines, i.e., IL-4, IL-5 and IL-13 as well as type 2 immune cells including alternatively activated macrophages, innate lymphoid cells and Th2 cells. However, the hallmark characteristic of filarial infections is a profound eosinophilia. Eosinophils are innate immune cells and pivotal in controlling helminth infections in general and filarial infections in particular. By modulating the function of other leukocytes, eosinophils support and drive type 2 immune responses. Moreover, as primary effector cells, eosinophils can directly attack filariae through the release of granules containing toxic cationic proteins with or without extracellular DNA traps. At the same time, eosinophils can be a driving force for filarial pathology as observed during tropical pulmonary eosinophilia in lymphatic filariasis, in dermatitis in onchocerciasis patients as well as adverse events after treatment of onchocerciasis patients with diethylcarbamazine. This review summarizes the latest findings of the importance of eosinophil effector functions including the role of eosinophil-derived proteins in controlling filarial infections and their impact on filarial pathology analyzing both human and experimental animal studies.

Litomosoides brasiliensis (Nematoda: Onchocercidae) infecting chiropterans in the Legal Amazon region, Brazil.

Chiropterans play an important role in the maintenance of the environmental balance, since they are pollinators, seed dispersers and predators. They contribute to transmission and spreading of microorganisms such as helminths, fungi, protozoa, bacteria and virus. The aim of the present study was to investigate natural filariid infection among bats in the Legal Amazon region, Brazil, by means of parasitological and molecular analyses. Blood samples were collected from 82 bats for blood smears and for DNA extraction via the polymerase chain reaction (PCR) assay. Microfilariae were observed in blood smears from Carollia perspicillata (2), Artibeus lituratus (1), Artibeus fimbriatus (2), Dermanura gnoma (2) and Glossophaga soricina (1). Five positive samples were detected through the PCR assay and four of these were also positive in blood smears. From genome sequencing and comparative analysis with sequences deposited in GenBank, one sample showed 99.31% similarity to the species Litomosoides brasiliensis. The present study expands the geographical distribution of L. brasiliensis, to include the state of Maranhão as an area of occurrence of this species and includes D. gnoma and A. fimbriatus as hosts in Brazil.

Zoonotic infection caused by Onchocerca japonica (Nematoda: Filarioidea) in a 69-year-old woman in Kanto Region, Eastern Honshu, Japan.

Reports of zoonotic infections caused by the filarial nematode Onchocerca japonica have recently increased in Japan. A 69-year-old woman living in Sosa City, Chiba Prefecture, Kanto Region, Honshu, developed a painful nodule at the metacarpophalangeal joint of the index finger of her right hand. The causative agent was identified as a female O. japonica based on the histopathological characteristics (i.e., cuticle with transverse triangular ridges but without inner striae) of the biopsy specimens of the nodule. The species identification was corroborated by cox1 gene sequencing of the worm tissues isolated from paraffin-embedded sections of the specimens. Subsequent to the excision of the nodule, followed by anthelmintic treatment, the patient remained asymptomatic. Human infection with O. japonica has not previously been reported in Kanto Region, Eastern Honshu. The present case is likely linked to the recent expansion of the geographic range of the Japanese wild boar into this area.

Design, synthesis, and in vitro evaluation of thiosemicarbazone derivatives as anti-filarial agents.

Effective macrofilaricidal drugs are not commercially available, and in an endeavour to find out new macrofilaricidal agents, in this research work, thiosemicarbazone derivatives have been prepared and tested against adult Setaria digitata, a cattle filarial parasite, as a model nematode for the filarial parasite, Wuchereria bancrofti. Lipinski and Veber rules have been used to design these molecules and found out that all the designed molecules show drug-like molecular properties. The in vitro anti-filarial potential of thiosemicarbazones against S. digitata was carried out using worm motility and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction colorimetric assays at 100 µg/ml concentration for the incubation period of 24 h. The standard drugs used at present for filaria, Albendazole, Ivermectin and Diethylcarbamazine were not able to kill the adult filarial worms effectively. In contrast, phenyl thiosemicarbazones with trifluoromethyl substitution at 3rd and 4th positions, 2-pyrrolyl, and isatinyl made the adult worms immotile and also showed 69%-83% inhibition in formazan formation an indicator of non viability.

ILC2s Control Microfilaremia During Litomosoides sigmodontis Infection in Rag2-/- Mice.

Group 2 innate lymphoid cells (ILC2s) are inducers of type 2 immune responses, but their role during filarial infection remains unclear. In the present study, we used the Litomosoides sigmodontis rodent model of filariasis to analyze ILC2s during infection in susceptible BALB/c mice that develop a chronic infection with microfilaremia and semi-susceptible C57BL/6 mice that eliminate the filariae shortly after the molt into adult worms and thus do not develop microfilaremia. ILC2s (CD45+ Lineage- TCRß- CD90.2+ Sca-1+ IL-33R+ GATA-3+) were analyzed in the pleural cavity, the site of L. sigmodontis infection, after the infective L3 larvae reached the pleural cavity (9 days post infection, dpi), after the molt into adult worms (30dpi) and during the peak of microfilaremia (70dpi). C57BL/6 mice had significantly increased ILC2 numbers compared to BALB/c mice at 30dpi, accompanied by substantially higher IL-5 and IL-13 levels, indicating a stronger type 2 immune response in C57BL/6 mice upon L. sigmodontis infection. At this time point the ILC2 numbers positively correlated with the worm burden in both mouse strains. ILC2s and GATA-3+ CD4+ T cells were the dominant source of IL-5 in L. sigmodontis-infected C57BL/6 mice with ILC2s showing a significantly higher IL-5 expression than CD4+ T cells. To investigate the importance of ILC2s during L. sigmodontis infection, ILC2s were depleted with anti-CD90.2 antibodies in T and B cell-deficient Rag2-/- C57BL/6 mice on 26-28dpi and the outcome of infection was compared to isotype controls. Rag2-/- mice were per se susceptible to L. sigmodontis infection with significantly higher worm burden than C57BL/6 mice and developed microfilaremia. Depletion of ILC2s did not result in an increased worm burden in Rag2-/- mice, but led to significantly higher microfilariae numbers compared to isotype controls. In conclusion, our data demonstrate that ILC2s are essentially involved in the control of microfilaremia in Rag2-/- C57BL/6 mice.

Genetic and morphological identification of filarial worm from Iberian hare in Portugal.

The Iberian hare (Lepus granatensis) is an endemic species of the Iberian Peninsula and the only hare species found in Portugal, although also being present in some areas of Spain. The reduction of wild hare populations due to several ecological and sanitary factors, has been raising growing concerns in the recent years. Despite different helminth species were already described in Iberian hares in Portugal, to this date, no filarial worms have been identified in this species. Furthermore, only a few studies on lagomorphs' onchocercid worms are available, referring to other hosts species of hares and/or rabbits. In this study, we describe the presence of filarial worms in the blood vessels of two adult Iberian hares collected in 2019 in continental Portugal. Morphology and sequencing data from the 12S rRNA, coxI, 18S rRNA, myoHC, hsp70 and rbp1 genes, showed that the filaroid species were genetically related with Micipsella numidica. However, the extension of the genetic differences found with M. numidica suggests that the filaroids specimens under study belong to a new species, that we provisionally named Micipsella iberica n. sp.. The body location of this putative new parasite species and its physiological implications indicate that it may constitute a potential menace to the already fragile Iberian hare justifying, therefore, further investigation regarding the morphological characterization, prevalence and real clinical impact of this new parasite in hares.

Cercopithifilaria spp. in ticks of companion animals from Asia: new putative hosts and vectors.

Cercopithifilaria bainae, Cercopithifilaria grassi, and Cercopithifilaria sp. II sensu Otranto et al., 2013 tick borne filarioids are typically found in dogs. Among them, Cercopithifilaria bainae has a worldwide distribution according to the occurrence of its tick vector, Rhipicephalus sanguineus sensu lato (s.l.). Nevertheless, in Asian countries, despite the wide presence of this tick species, data on Cercopithifilaria spp. are scant. Therefore, this study aimed to assess the occurrence of these dermal filarioids in ixodid ticks collected on dogs and cats from Asian countries, providing a better epidemiological picture on their distribution in this continent. Ticks (n = 687) of the species Rhipicephalus sanguineus s. l. (n = 667), Rhipicephalus haemaphysaloides (n = 8), Haemaphysalis longicornis (n = 7), Haemaphysalis campanulata (n = 1), Haemaphysalis wellingtoni (n = 2), Haemaphysalis hystricis (n = 1), and Ixodes sp. (n = 1) were collected on dogs and cats under the frame of previous studies in China, India, Indonesia, Malaysia, Singapore, Taiwan, Thailand, the Philippines and Vietnam. Tick samples were molecularly screened for Cercopithifilaria spp. by conventional PCR and real-time PCR using two pair of primers targeting partial sequences of cytochrome c oxidase 1 gene. Overall, Cercophitifilaria spp. DNA was detected in 9.5% (n = 65/687) of the tick specimens tested, with C. bainae being the most prevalent species (8.9%), followed by C. grassii (0.6%). Most Cercophitifilaria spp. positive ticks were collected on dogs (92.3%; 60/65); whereas ticks collected on cats represented 7.7% of the positive specimens. In addition, Cercopithifilaria spp. were mostly detected in R. sanguineus s.l. ticks (96.9%; 63/65), followed by Rhipicephalus haemaphysaloides (3.1%; 2/65). Data herein presented demonstrate the occurrence of dermal tick borne filarioids of the genus Cercopithifilaria in several Asian countries, with C. bainae being the most prevalent species. We also report for the first time the molecular detection of C. bainae in R. sanguineus s.l. ticks collected on cats, as well as in R. haemaphysaloides ticks, suggesting that the biological cycle of this filarioid species may involve other intermediate and definitive hosts than R. sanguineus s.l. and dogs. However, confirmatory studies on the role of other tick species and domestic cats on the biology of C. bainae are advocated.

Canine filaria species in selected lymphatic filariasis endemic and non-endemic areas in Sri Lanka.

Subperiodic brugian filariasis and dirofilariasis show a rising trend in Sri Lanka posing a threat to public health. As information was limited on canine filaria species in Sri Lanka, we studied the filaria parasites among dog populations in lymphatic filariasis (LF) endemic and non-endemic regions by microscopy and molecular methods. Thick blood smears (TBSs) were performed among 295 dogs presenting to veterinary clinics for surgical or sterilization procedures in Galle (LF endemic) and Mullaitivu (LF non-endemic) districts, of which 55.6% were positive for any microfilariae. We identified Dirofilaria repens (50.8%) and Brugia spp. (20.6%) by microscopy, which, included mono-infections (D. repens 35.3% and Brugia spp. 5%) and co-infections (15.6%). Infections in Galle and Mullaitivu were 61% and 44.9% respectively. The brugian filariasis rate was significantly higher among canines in LF endemic Galle district (29.9%) than in Mullaitivu (LF non-endemic) (1.1%) (P < 0.001), while D. repens infections were comparable in both districts. Genomic DNA extracted from 10% of microfilariae positive TBSs was amplified using pan-filarial primers targeting the internal-transcriber-spacer region-2 (ITS-2). Sequencing of amplicons confirmed the presence of D. repens (89.28%), Brugia pahangi (7.14%) and B. malayi (3.57%) infections. The phylogeny constructed and analysed in MEGA X indicated genetic variability among D. repens and B. pahangi isolates from Sri Lanka. With this study, we were able to report B. pahangi infections for the first time in Sri Lanka.