Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Historical biogeography among species of Varestrongylus lungworms (Nematoda: Protostrongylidae) in ungulates: episodic expansion and host colonization linking Eurasia and North America.

Varestrongylus lungworms (Nematoda: Protostrongylidae) include 10 nominal species that parasitize wild and domesticated artiodactyles. Eight species are endemic to the western Palearctic and Eurasia, whereas two are limited in distribution to the Nearctic. Complex host associations, primarily among Cervidae and Bovidae (Caprinae), and biogeography were explored based on direct comparisons of parasite and host phylogenies to reveal the historical development of this fauna. Diversification among Varestrongylus species has an intricate history extending over the Pliocene and Quaternary involving episodic processes for geographic and host colonization: (1) Varestrongylus has origins in Eurasia with independent expansion events into bordering ecozones; (2) cervids are ancestral hosts; (3) the caprine-associated V. pneumonicus is basal and a result of an independent host colonization event; (4) secondary diversification, linked to sequential and independent host colonization events, occurred within cervids (V. sagittatus + V. tuvae; V. alpenae; and V. capreoli, V. alces + V. eleguneniensis); (5) at least two additional host colonization events into caprines occurred, followed or not by diversification (V. qinghaiensis + V. longispiculatus; V. capricola, respectively); (6) two independent events of geographic expansion into North America from Eurasia with cervids in the late Pliocene and early Pleistocene are postulated (V. alpenae, V. eleguneniensis). Comparisons based on phylogenetic hypotheses derived from comparative morphology and molecular inference for these nematodes are consistent with the postulated history for coevolutionary and biogeographic history. Episodes of geographic and host colonization, often in relation to rapid shifts in climate and habitat perturbation, have dominated the history of diversification of Varestrongylus.

Varestrongylus (Nematoda: Protostrongylidae), lungworms of ungulates: a phylogenetic framework based on comparative morphology.

Varestrongylus Bhalerao, 1932 comprises ten valid lungworm species infecting wild and domestic ungulates from Eurasia and North America. Here, we present a phylogenetic hypothesis for the genus based on morphological characters in a broader context for the family Protostrongylidae and discuss species relationships and aspects of character evolution. Phylogenetic analysis of 25 structural attributes, including binary and multistate characters, among the 10 species of Varestrongylus resulted in one fully resolved most parsimonious tree (61 steps; consistency index = 0.672, retention index = 0.722, and consistency index excluding uninformative characters = 0.667). Varestrongylus forms a monophyletic clade and is the sister of Pneumostrongylus, supporting recognition of the subfamily Varestrongylinae. Monophyly for Varestrongylus is diagnosed by six unequivocal synapomorphies, all associated with structural characters of the copulatory system of males. Varestrongylus pneumonicus is basal, and sister to all other species within the genus, which form two subclades. The subclade I contains V. sagittatus + V. tuvae and V. qinghaiensis + V. longispiculatus. Subclade II contains V. alpenae, V. capricola, V. capreoli, and V. eleguneniensis + V. alces. Both subclades are diagnosed by two unambiguous synapomorphies. Highlighted is the continuing importance of phylogenetic assessments based on comparative morphology as a foundation to explore the structure of the biosphere across space and time.

The cockroach Periplaneta americana as a potential paratenic host of the lungworm Aelurostrongylus abstrusus.

INTRODUCTION: Aelurostrongylus abstrusus is a well-known nematode affecting the respiratory system of felids worldwide. Snails and slugs act as intermediate hosts of this parasite, whereas rodents, birds and reptiles may serve as paratenic hosts. Periplaneta americana, the American brown cockroach, shares the same habitat and ecological features (e.g. nocturnal activity) with both snails and cats. The aim of this study was to evaluate the capability of P. americana to maintain alive A. abstrusus third stage larvae (L3s) after artificial inoculation. MATERIAL AND METHODS: Twenty-five specimens of P. americana were infected with 100 A. abstrusus L3s collected from experimentally infected Cornu aspersum snails, whereas five specimens were used as control group. After the infection, cockroaches were maintained in individual plastic boxes until dissection for the presence of L3s at 1 (T1), 5 (T5), 10 (T10), 15 (T15), and 20 (T20) days post-infection. RESULTS: Except for T15, alive A. abstrusus L3s (n = 63) were found at all time-points, being 26, 19, 16 and 2 L3s retrieved at T1, T5, T10 and T20, respectively. Eleven (17.4%) L3s were found within the digestive tract, 10 (15.9%) in other-than-digestive organs and 42 (66.7%) in the exoskeleton and associated tissues. Nine out of the twenty-five experimentally inoculated cockroaches (36%) died soon after the artificial infection (T1), while in the control group, two out of the five (40%) died before the end of the study (T15) with no difference in the mortality rate between groups. DISCUSSION: Results of this study suggest that P. americana could act as a paratenic host of A. abstrusus. Periplaneta americana cockroaches, have a ubiquitous distribution and may be preyed by cats, representing a potential source of infection to cats living in endemic areas.

Molecular phylogeny of the Pseudaliidae (Nematoda) and the origin of associations between lungworms and marine mammals.

Metastrongyloid lungworms from the family Pseudaliidae infect the lungs and cranial sinuses of cetaceans worldwide, except Stenuroides herpestis, which exhibits a striking terrestrial association with the Egyptian mongoose, Herpestes ichneumon. Previous phylogenies of the Metastrongyloidea that included some (2-7) marine species of the Pseudaliidae confirmed that these species are closely related, but also grouped species of Parafilaroides (family Filaroididae) between the members of the Pseudaliidae. In this study we extracted DNA from representatives of all six genera of the Pseudaliidae and amplified the second internal transcribed spacer (ITS2) and cytochrome c oxidase subunit I (cox1) genes in order to investigate the concept of the Pseudaliidae as a monophyletic unit. Three species of Parafilaroides were also included in the analysis. Maximum Likelihood and Bayesian Inference analyses of the concatenated genes resulted in a well-supported clade including the marine pseudaliids, S. herpestis and Parafilaroides spp. These findings validate the status of S. herpestis as a pseudaliid species and support the inclusion of Parafilaroides in the Pseudaliidae. Although males of Parafilaroides spp. lack a copulatory bursa, this trait itself is highly variable in the Pseudaliidae, which include abursate species. Furthermore, life cycles seem to be closely similar between both taxa. When all the available phylogenetic data on the Metastrongyloidea were mapped of onto a phylogeny of the Laurasiatheria, there was a strong suggestion that the Pseudaliidae may have descended from ancestors infecting terrestrial carnivores, with odontocetes being colonized in the marine realm as a result of a host-switching event from pinnipeds through shared fish prey. The origin of the association between S. herpestis and mongooses remains uncertain.

The Prevalence and Control of Lungworms of Pastoral Ruminants in Iran.

Lungworms of the genera Dictyocaulus, Muellerius, Protostrongylus, and Cystocaulus are common helminths of domestic and wild ruminants with substantial veterinary and economic importance. Several studies have assessed the presence and prevalence of lungworm infections in ruminants in Iran. This report compiles the available scientific information about the occurrence of lungworms in domestic and wild ruminants in Iran between 1931 and June 2022 to give an insight into their epidemiology, and where possible to describe drug treatment efficacy. For this purpose, national and international scientific databases were searched. Overall, 54 publications comprising 33 articles in peer-reviewed journals, 8 conference papers, and 13 dissertations were evaluated regarding prevalence data; and an additional 4 peer-reviewed articles were evaluated regarding drug efficacy. Seven species of lungworms, namely Dictyocaulus filaria, Dictyocaulus viviparus, Dictyocaulus eckerti, Protostrongylus rufescens, Protostrongylus raillietti, Muellerius capillaris, and Cystocaulus ocreatus have been recorded from different ruminant hosts in Iran. Thirty-three studies conducted on small ruminant (sheep and goat) lungworms reported prevalences of lungworm infection of 11.6%, 45.81% and 66.29% using abattoir meat inspection, Baermann technique and fecal flotation, respectively. Eight studies conducted on large ruminants (cattle and water buffalo) reported prevalences of infection of 14.83%, 13.98% and 5% using abattoir meat inspection, the Baermann technique and fecal flotation, respectively. The prevalence of infection in wild ruminants was variable across examined species; 38% in urial, 37% in wild goats, 5% in goitered gazelles and 67% in red deer, in addition to a single case report in roe deer. There are few contemporary studies assessing the efficacy of currently available broad-spectrum anthelmintic compounds against lungworms in Iran. The high prevalence of multiple lungworm species in Iran, combined with a lack of information about drug efficacy, supports the need to improve the understanding of these important nematode parasites and inform the development of sustainable control strategies. The aim of this review and meta-analysis is to provide a baseline for future conventional parasitology and next generation molecular epidemiological studies of lungworm infection in pastoral ruminants in Iran.

PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

The giant African snail Achatina (Lissachatina) fulica Bowdich, 1822 as an intermediate host of Aelurostrongylus abstrusus (Railliet, 1898) in the Rio de Janeiro state, Brazil.

Aelurostrongylus abstrusus is a metastrongilid nematode that causes feline aelurostrongylosis. The adult life stage parasitizes the lungs of wild and domestic felids. Terrestrial mollusks act as its intermediate host, and birds, amphibians, and small mammals act as paratenic hosts. Aelurostrongylus abstrusus presents a worldwide distribution, and in Brazil it has been recorded in most states. The present study investigates the occurrence of Aelurostrongylus abstrusus in terrestrial mollusks from urban areas of two out of the six Mesoregions of Rio de Janeiro state: Metropolitana and Centro Fluminense (46 municipalities). Larvae were recovered from the tissue of the mollusks collected during the surveys and identified based on morphology and sequences of the Internal Transcribed Spacer 2 (ITS2). In all, 2791 mollusks, belonging to 14 different species, were collected. Larvae of A. abstrusus were recovered from two species: Achatina fulica in 22 out of 46 municipalities, and from the slug Latipes erinaceus in 1 out of 46 municipalities. Aelurostrongylus abstrusus was recorded in half (50%) of the municipalities of the Metropolitana Mesoregion and in 44% of those of the Centro Fluminense Mesoregion. Results indicate a strong association between A. abstrusus and A. fulica, since 99% of the mollusks infected with A. abstrusus were A. fulica. This study recorded A. abstrusus in the Centro Fluminense Mesoregion and naturally parasitizing L. erinaceus for the first time. Despite the high number of mollusks infected with A. abstrusus and their wide distribution in Rio de Janeiro State, there are still few reports of infection in Brazilian felids, probably given that this is an under-diagnosed parasitosis in this country. Further studies in other regions of the Rio de Janeiro State, and other Brazilian States, focusing on intermediate and definitive hosts, particularly in the urban environments where they coexist, are needed. These studies will enable further understanding of the epidemiology and prevention of aelurostrongylosis.

Gurltia paralysans: A Neglected Angio-Neurotropic Parasite of Domestic Cats (Felis catus) and Free-Ranging Wild Felids (Leopardus spp.) in South America.

Gurltia paralysans is a neglected and re-emerging metastrongyloid angio-neurotropic nematode causing severe chronic meningomyelitis in domestic cats (Felis catus) as well as in free-ranging small wild felids such as kodkods (Leopardus guigna), margays (Leopardus wiedii) and the northern tiger cat (Leopardus triginus) in South America. Within these definitive hosts (DH), adult males and females of G. paralysans parasitize the leptomeningeal veins of the subarachnoid space and/or the meningeal veins of spinal cord parenchyma, inducing vascular alterations. Feline gurltiosis has been associated with progressive thrombophlebitis of the meningeal veins, resulting in ambulatory paraparesis, paraplegia, ataxia, hindlimb proprioceptive deficit, uni- or bilateral hyperactive patellar reflexes, faecal and urinary incontinence, and tail paralysis. The complete life cycle of G. paralysans has not been elucidated yet, but most probably involves gastropods as obligate intermediate hosts (IH). In terms of epidemiology, G. paralysans infections in domestic and wild felids are scattered around various South American countries, with hyperendemic areas in southern parts of Chile. Etiological diagnosis of G. paralysans still represents a challenge for clinicians due to a lack of evidence of the excretion of either eggs or larvae in faeces or in other body fluids. Diagnosis is based on clinical neurological signs, imaging findings through computed tomography (CT), myelography, magnetic resonance imaging (MRI), and post mortem examination. Nonetheless, novel diagnostic tools have been developed, including semi-nested PCR for detecting circulating G. paralysans DNA in the cerebrospinal fluid, serum and blood samples as well as in serological diagnostic kits detecting parasite-derived antigens, but these need validation for routine usage. The hypothetical life cycle of G. paralysans is addressed in this article, including the exogenous stages (i.e., eggs, and first- (L1), second- (L2) and third-stage (L3) larvae) and obligate gastropod IH and/or paratenic hosts (PH), and we propose possible anatomical migration routes of infective L3 that reach the leptomeningeal veins in vivo. Finally, the pro-inflammatory endothelium- and leukocyte-derived innate immune reactions of the host against G. paralysans, which most likely result in thrombophlebitis and meningomyelitis, are briefly touched on.

A Molecular Survey on Neglected Gurltia paralysans and Aelurostrongylus abstrusus Infections in Domestic Cats (Felis catus) from Southern Chile.

Gurltia paralysans and Aelurostrongylus abstrusus are neglected metastrongyloid nematode species which infect domestic and wild cats in South American countries and in Chile, but no epidemiological studies on concomitant infections have been conducted in Chile so far. The aim of this study was not only to evaluate the occurrence of concomitant infections, but also to identify epidemiological risk factors associated with of G. paralysans and A. abstrusus infections in urban domestic cats (Felis catus) from Southern Chile. Blood samples from clinically healthy domestic cats from three cities of Southern Chile-Temuco, Valdivia, and Puerto Montt-were analyzed by an experimental semi-nested PCR protocol. A total of 171 apparently healthy domestic cats in Temuco (n = 68), Valdivia (n = 50), and Puerto Montt (n = 53) were sampled and analyzed. A total of 93 domestic cats (54.4%) were positive for G. paralysans, and 34 (19.9%) were positive for A. abstrusus infections. From those animals, 34 (19.9%) were co-infected. Cats positive with G. paralysans were found in all three cities; 47.2% in Puerto Montt, 48% in Valdivia, and 64.7% in Temuco. Levels of infection for A. abstrusus in the population under study were 4% (Valdivia), 10% (Puerto Montt), and 32.4% (Temuco). The present large-scale epidemiological study confirmed the presence of these neglected nematodes in domestic cat populations in Southern Chile, and described the possible risk factors associated with feline gurltiosis and aelurostrongylosis.

The biogeography of the caribou lungworm, Varestrongylus eleguneniensis (Nematoda: Protostrongylidae) across northern North America.

Varestrongylus eleguneniensis (Nematoda; Protostrongylidae) is a recently described species of lungworm that infects caribou (Rangifer tarandus), muskoxen (Ovibos moschatus) and moose (Alces americanus) across northern North America. Herein we explore the geographic distribution of V. eleguneniensis through geographically extensive sampling and discuss the biogeography of this multi-host parasite. We analyzed fecal samples of three caribou subspecies (n = 1485), two muskox subspecies (n = 159), and two moose subspecies (n = 264) from across northern North America. Protostrongylid dorsal-spined larvae (DSL) were found in 23.8%, 73.6%, and 4.2% of these ungulates, respectively. A portion of recovered DSL were identified by genetic analyses of the ITS-2 region of the nuclear rDNA or the cytochrome oxidase c subunit I (COI) region of the mtDNA. We found V. eleguneniensis widely distributed among caribou and muskox populations across most of their geographic prange in North America but it was rare in moose. Parelaphostrongylus andersoni was present in caribou and moose and we provide new geographic records for this species. This study provides a substantial expansion of the knowledge defining the current distribution and biogeography of protostrongylid nematodes in northern ungulates. Insights about the host and geographic range of V. eleguneniensis can serve as a geographically extensive baseline for monitoring current distribution and in anticipating future biogeographic scenarios under a regime of accelerating climate and anthropogenic perturbation.

Validity of genus Perostrongylus Schlegel, 1934 with new data on Perostrongylus falciformis (Schlegel, 1933) in European badgers, Meles meles (Linnaeus, 1758): distribution, life-cycle and pathology.

BACKGROUND: A century of debates on the taxonomy of members of the Metastrongyloidea Molin, 1861 led to many reclassifications. Considering the inconstant genus assignation and lack of genetic data, the main aim of this study was to support the validity of the genus Perostrongylus Schlegel, 1934, previously considered a synonym of Aelurostrongylus Cameron, 1927, based on new molecular phylogenetic data and to understand its evolutionary relationships with other metastrongyloid nematodes. RESULTS: Specimens of lungworm collected from European badgers in Germany, Romania and Bosnia and Herzegovina were morphologically and molecularly (rDNA, cox1) characterized. From a phylogenetic standpoint, Perostrongylus is grouped with high support together with the genera Filaroides van Beneden, 1858 and Parafilaroides Dougherty, 1946 and includes probably two species: Perostrongylus falciformis (Schlegel, 1933), a parasite of Meles meles in Europe and P. pridhami (Anderson, 1962), a parasite of Neovison vison in North America. Perostrongylus and Aelurostrongylus are assigned to different clades. Aelurostrongylus becomes a monotypic genus, with the only species Aelurostrongylus abstrusus (Railliet, 1898). In addition, we provide morphological and morphometric data for the first-stage (L1), second-stage (L2), and third-stage (L3) larvae of P. falciformis and describe their development in experimentally infected Cornu aspersum snails. The pathological and histopathological lesions in lungs of infected European badgers are also described. This is the first record of P. falciformis in Romania. CONCLUSIONS: Molecular phylogenetic and morphological data support the validity of the genus Perostrongylus, most probably with two species, P. falciformis in European badgers and P. pridhami in minks in North America. The two genera clearly belong to two different clades: Perostrongylus is grouped together with the genera Filaroides and Parafilaroides (both in the family Filaroididae Schulz, 1951), whereas Aelurostrongylus belongs to a clade with no sister groups.

Angiostrongylus vasorum and Aelurostrongylus abstrusus: Neglected and underestimated parasites in South America.

The gastropod-borne nematodes Angiostrongylus vasorum and Aelurostrongylus abstrusus are global causes of cardio/pulmonary diseases in dogs and cats. In the last decade, the number of reports on canine and feline lungworms has increased in several areas of Europe and North America. The unspecific clinical signs and prolonged course of these diseases often renders diagnosis challenging. Both infections are considered as emerging and underestimated causes of disease in domestic pets. In South America, little information is available on these diseases, apart from occasional reports proving the principle presence of A. vasorum and A. abstrusus. Thus, the purpose of this review is to summarize reports on infections in both domestic and wildlife animals in South America and to increase the awareness on gastropod-borne metastrongyloid parasites, which also include important zoonotic species, such as A. cantonensis and A. costaricensis. This review highlights the usefulness of diagnostic tools, such as the Baermann funnel technique, serology and PCR, and proposes to include these routinely on cases with clinical suspicion for lungworm infections. Future national epidemiological surveys are recommended to be conducted to gain a deeper insight into the actual epidemiological situation of gastropod-borne parasitoses in South America.

Prevalence of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Crenosoma vulpis larvae in native slug populations in Germany.

Metastrongyloid parasites represent sparsely studied parasites of dogs and cats in Germany. Recent European surveys indicate that these parasites are spreading in Europe. Actual data on prevalence of Angiostrongylus vasorum in dogs and foxes reveal several endemic foci in Germany. However, actual data on the prevalence of A. vasorum and other metastrongyloid lungworm larvae in a wide range of slug and snail intermediate hosts, such as Arion lusitanicus, are missing for Germany. To fill this gap, we conducted an epidemiological survey on native German slugs in selected regions of Hesse and Rhineland-Palatinate. The focus was on slugs, because in study areas slugs appear to be more abundant than snails. Slugs were collected throughout different seasons of the year in areas that were previously proven to be hyperendemic for A. vasorum fox infections. Overall, a total of 2701 slugs were collected and examined for lungworm larvae via artificial digestion. The number of A. vasorum larvae per slug varied considerably (1-546 larvae per specimen). Some hotspot areas with high A. vasorum prevalence in slugs (up to 19.4%) were identified. The overall A. vasorum prevalence varied with season with largest number of slugs infected in summer (9.1%) and lowest number in winter (0.8%). The current study revealed a total A. vasorum prevalence of 4.7% in slugs based on microscopic analyses. Confirmation of lungworm species was made by specific duplex-real-time PCRs. Hence, these data demonstrate that final hosts are at a permanent risk for A. vasorum infections during all seasons when living in investigated areas. Besides A. vasorum, other lungworm larvae were also detected, such as Crenosoma vulpis (the fox lungworm, 2.3%) and Aelurostrongylus abstrusus (feline lungworm, 0.2%).

Natural infection of the feline lungworm Aelurostrongylus abstrusus in the invasive snail Achatina fulica from Argentina.

The giant African snail Achatina fulica is an invasive mollusk native to Africa, the first record in Argentina was in Puerto Iguazú, in northeastern Argentina in 2010. Recently it was reported in Corrientes Province. This snail can act as an intermediate host of Metastrongyloidea nematodes of importance in public health as: Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus vasorum. Taking into account the presence of A. fulica in Argentina, the objectives of this study is to assess the presence of Metastrongyloidea nematodes in this mollusk species in Puerto Iguazú, Misiones, close to the international border with Brazil and Paraguay. A total of 451 samples were collected from February 2014 to November 2015. The snails were processed using a digestion technique to recover the parasites. A total of 206 nematodes larvae were founded in the digestion solution of 10 hosts (P=2%; MA=0.5; MI=21). Third larval stage (L3) nematodes identified as Aelurostrongylus abstrusus were founded parasitizing the snails. No other larval stage was observed. This species has veterinary importance because it causes 'aelurostrongilosis', also known as feline strongyloidosis. This study constitutes the first record of a Metastrongyloidea nematode in A. fulica in Argentina and also highlights the susceptibility of this mollusk as intermediate host of other helminthes of health importance. The present study suggests that there is a need to establish an epidemiological monitoring system in order to prevent the possible installation of an infected mollusks focus.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

BACKGROUND: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. RESULTS: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. CONCLUSIONS: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Complete mitochondrial genome of Angiostrongylus malaysiensis lungworm and molecular phylogeny of Metastrongyloid nematodes.

Angiostrongylus malaysiensis is a nematode parasite of various rat species. When first documented in Malaysia, it was referred to as A. cantonensis. Unlike A. cantonensis, the complete mitochondrial genome of A. malaysiensis has not been documented. We report here its complete mitogenome, its differentiation from A. cantonensis, and the phylogenetic relationships with its congeners and other Metastrongyloid taxa. The whole mitogenome of A. malaysiensis had a total length of 13,516bp, comprising 36 genes (12 PCGs, 2 rRNA and 22 tRNA genes) and a control region. It is longer than that of A. cantonensis (13,509bp). Its control region had a long poly T-stretch of 12bp which was not present in A. cantonensis. A. malaysiensis and A. cantonensis had identical start codon for the 12 PCGs, but four PCGs (atp6, cob, nad2, nad6) had different stop codon. The cloverleaf structure for the 22 tRNAs was similar in A. malaysiensis and A. cantonensis except the TΨC-arm was absent in trnV for A. malaysiensis but present in A. cantonensis. The Angiostrongylus genus was monophyletic, with A. malaysiensis and A. cantonensis forming a distinct lineage from that of A. costaricensis and A. vasorum. The genetic distance between A. malaysiensis and A. cantonensis was p=11.9% based on 12 PCGs, p=9.5% based on 2 rRNA genes, and p=11.6% based on 14 mt-genes. The mitogenome will prove useful for studies on phylogenetics and systematics of Angiostrongylus lungworms and other Metastrongyloid nematodes.

Angiostrongylus vasorum in the eye: new case reports and a review of the literature.

BACKGROUND: Nematodes of the genus Angiostrongylus are important causes of potentially life-threatening diseases in several animal species and humans. Angiostrongylus vasorum affects the right ventricle of the heart and the pulmonary arteries in dogs, red foxes and other carnivores. The diagnosis of canine angiostrongylosis may be challenging due to the wide spectrum of clinical signs. Ocular manifestations have been seldom reported but have serious implications for patients. METHODS: The clinical history of three cases of infection with A. vasorum in dogs diagnosed in UK, France and Italy, was obtained from clinical records provided by the veterinary surgeons along with information on the diagnostic procedures and treatment. Nematodes collected from the eyes of infected dogs were morphologically identified to the species level and molecularly analysed by the amplification of the nuclear 18S rRNA gene. RESULTS: On admission, the dogs were presented with various degrees of ocular discomfort and hyphema because of the presence of a motile object in the eye. The three patients had ocular surgery during which nematodes were removed and subsequently morphologically and molecularly identified as two adult males and one female of A. vasorum. CONCLUSIONS: Three new cases of canine ocular angiostrongylosis are reported along with a review of other published clinical cases to improve the diagnosis and provide clinical recommendation for this parasitic condition. In addition, the significance of migratory patterns of larvae inside the host body is discussed. Veterinary healthcare workers should include canine angiostrongylosis in the differential diagnosis of ocular diseases.

Development of Crenosoma vulpis in the common garden snail Cornu aspersum: implications for epidemiological studies.

BACKGROUND: Crenosoma vulpis (Dujardin, 1845), the fox lungworm, is a metastrongyloid affecting the respiratory tract of red foxes (Vulpes vulpes), dogs (Canis familiaris) and badgers (Meles meles) living in Europe and North America. The scant data available on the intermediate hosts of C. vulpis, as well as the limited information about the morphology of the larvae may jeopardise epidemiological studies on this parasite. METHODS: Suitability and developmental time of C. vulpis in the common garden snail Cornu aspersum (= Helix aspersa) was assessed at selected days post-infection (i.e. 3, 6, 10, 15, 20 and 180). Nematodes were preserved in 70% ethanol, cleared and examined as temporary mounts in glycerol for morphological descriptions of first- and third-stage larvae. In addition, nematodes collected from the dog and the experimentally infected snails were molecularly analysed by the amplification of the nuclear 18S rRNA gene. RESULTS: Specimens of C. aspersum digested before the infection (n = 10) were negative for helminth infections. Out of 115 larvae recovered from infected gastropods (mean of 9.58 larvae per snail), 36 (31.3%) were localised in the foot and 79 (68.7%) in the viscera. The 18S rDNA sequences obtained from larvae collected from the dog and the snail tissues displayed 100% identity to the nucleotide sequence of C. vulpis. CONCLUSIONS: Cornu aspersum is herein reported for the first time as a suitable intermediate host of C. vulpis. This snail species may play an important role for the infection of animals living in regions of the Mediterranean basin. In addition, this study provides more details on the morphological descriptions of L1 and L3 and supports future investigations on the epidemiology of this little known parasite.

Guide to the Parasites of Fishes of Canada Part V: Nematoda.

Keys are provided for the identification of the nematode species known to be parasites of Canadian fishes. The nematodes are described and illustrated, with a note of the site(s) they occupy in named fish host(s) and their geographical distribution. Parasite records are given by author and date, full details of which can be found in a bibliography of over 800 references. Diagnoses and keys for 22 Families, 47 genera and 88 species of nematodes are also given, together with a glossary of terms, a host-parasite list, and indices to both nematode parasites and hosts.