Feline lungworms in Greece: copromicroscopic, molecular and serological study.

Aelurostrongylus abstrusus (Nematoda, Metastrongyloidea) causes verminous pneumonia in cats worldwide. This study evaluated the seroprevalence of A. abstrusus antibodies in 220 stray and free-roaming cats from insular (Mykonos, Crete, Skopelos) and continental (Thessaloniki, Attica) Greece. The results were compared with morphological and biomolecular identification of first-stage larvae (L1) in faeces. Positive cats were observed in all 5 areas: 13/97 (13.4%), 1/32 (3.1%), 7/26 (26.9%), 3/18 (16.7%) and 5/47 (10.6%) cats tested positive for A. abstrusus L1 by Baermann examination, and 33/97 (34.0%), 7/32 (21.9%), 6/26 (23.1%), 3/18 (16.7%) and 11/47 (23.4%) were seropositive, in Mykonos, Crete, Skopelos, Thessaloniki and Attica, respectively. Troglostrongylus brevior L1 were found in 12/97 (12.4%), 3/26 (11.5%) and 2/47 (4.3%) cats from Mykonos, Skopelos and Attica respectively. Six of the 220 cats (2.7%), i.e. 4/97 (4.1%) from Mykonos and 2/26 (7.7%) from Skopelos, shed L1 of both A. abstrusus and T. brevior. Sixty samples were ELISA-positive (27.3%, 95% CI: 21.5-33.7%), of which 21 (35%) tested copromicroscopically positive (19 monospecific infections and 2 mixed with Troglostrongylus brevior), and 5 were positive for T. brevior L1 only. Among seronegative cats (n = 140), L1 of A. abstrusus were additionally detected in 8 (5.7% out of 140) cats (i.e. 4 monospecific infections and 4 mixed with T. brevior), and in 6 (4.3% out of 140) cats, L1 of T. brevior as monospecific infection were detected. This study confirms the presence of lungworms in Greece and suggests that the number of cats infected with/exposed to metastrongylids is higher than detected by faecal examinations.

Surprisingly long body length of the lungworm Parafilaroides gymnurus from common seals of the Dutch North Sea.

Lungworms of the genera Parafilaroides and Otostrongylus are responsible for parasitic bronchopneumonia, the foremost disease of eastern Atlantic common seals (EACS, Phoca vitulina vitulina) in the Dutch North Sea. Recently, there have been increased reports of lungworm cases and observations of unusually long Parafilaroides sp. adults in this location. The initial aim of this study was to confirm the identity of the Parafilaroides species infecting this population. Parafilaroides are usually small and delicate, making them difficult to extract from host tissue, and there is often difficulty accessing fresh specimens for morphological study. The large size of the Dutch worms and the accessibility of specimens from numerous animals enabled the description and measurement of many intact specimens (N = 64) from multiple host animals (N = 20). Species identity was confirmed by targeted sequencing of ribosomal and mitochondrial DNA amplicons from a subset of worms. Worm morphology was consistent with descriptions for P. gymnurus, but the mature females were 1.9-fold and 3.4-fold longer than those recovered from French EACS (P ≤ 0.001) and Canadian western Atlantic common seals (Phoca vitulina concolor; P ≤ 0.0001). They were also significantly longer than mature female P. gymnurus described from other seal species, with the exception of those from harp seals of Les Escoumins, Quebec. We suggest that intraspecific genetic differences in P. gymnurus and the environment within the host could contribute to the variation reported here. This study is the first to describe P. gymnurus using morphological and molecular methods and should serve as a reference for identification of the species.

The lungworm, Halocercus brasiliensis (Nematoda: Pseudaliidae), from Guiana Dolphins Sotalia guianensis from Brazil with Pathological Findings.

The Guiana dolphin, Sotalia guianensis, is a small cetacean species found on the Atlantic coast of Central and South America from Honduras to Santa Catarina State, Brazil. The nematode Halocercus brasiliensis infects this cetacean, resulting in lung pathologies and death. The present study aimed to conduct a morphological and morphometric study of specimens of H. brasiliensis collected from S. guianensis from the coast of the state of Espírito Santo, Brazil. For this study, 7 dolphins were collected and examined for the presence of lung parasites. Nematodes were collected and processed for light microscopy, and lung fragments were processed for histological analysis. The nematodes were identified as H. brasiliensis according to their morphology and morphometry. The histopathological analysis revealed mineralization of the bronchiolar cartilage and inflammatory process. The parasitic infections by H. brasiliensis in S. guianensis can contribute to the debilitating status of these cetaceans, resulting in their stranding and accidental capture in fishing nets.

Severe Verminous Pneumonia Caused by Natural Mixed Infection with Aelurostrongylus abstrusus and Angiostrongylus chabaudi in a European Wildcat from Western Balkan Area.

AIMS: Aelurostrongylus abstrusus (Railliet, 1898) and Angiostrongylus chabaudi (Biocca, 1957) are important cardiopulmonary metastrongyloids in felids. This case report describes, for the first time, a natural and patent mixed infection caused by A. abstrusus and A. chabaudi in a European wildcat (Felis silvestris silvestris) from Bosnia and Herzegovina. Most important, this is a rare report of fatal parasitism in wildlife, involving a severe verminous bronchopneumonia and gastrointestinal nematode and cestode infection with Toxocara cati, Taenia taeniaeformis, Aonchotheca putorii, and Ancylostoma spp. RESULTS: Emphasis is set to detailed description of granulomatous, interstitial verminous bronchopneumonia, morphological description of A. abstrusus and A. chabaudi, and molecular confirmation of diagnosis by triplex PCR. CONCLUSION: The data provided in this study contribute to the knowledge on the epizootiology and pathological effect of these neglected metastrongyloids in European wildcat.

Identification of first-stage dorsal-spined lungworm larvae of Tunisian barbary red deer: First report of Varestrongylus sagittatus and Elaphostrongylus cervi in Africa.

Barbary red deer (Cervus elaphus barbarus) is a protected rare subspecies of red deer. The study of its Protostrongylidae fauna based only on sporadic necropsy of naturally dead animals is difficult. Therefore diagnosis of lungworms rely mainly on the identification of the first stage larvae (L1). The L1 of the different species are not readily diagnosed on morphological basis since much variation is recorded within and among dorsal-spined larvae belonging to various species. The aim of this study was to identify the dorsal-spined lungworm larvae of the Barbary red deer. A discriminant function was established, using the measurements of L1 lungworms recorded from red deer in the literature, then applied to identify 220 dorsal-spined larvae extracted from 25 Tunisian Barbary red deer fresh fecal samples. Also the ITS2 region of rDNA of four pools of larvae (n = 25-60) were amplified, sequenced and analyzed. Using discriminant analysis of morphological traits, Elaphostrongylus cervi and Varestongylus sagittatus were identified. Molecular identification confirmed the presence of E. cervi which is the most prevalent species. This study represents the first identification of V. sagittatus and E. cervi in North Africa.

Larval development of Aelurostrongylus abstrusus in experimentally infected Rumina decollata snails.

Aelurostrongylus abstrusus is a lungworm distributed worldwide that affects wild and domestic cats, causing bronchopneumonia of varying intensity. Snails serve as intermediate hosts. The aim of the present study was to assess the larval development of A. abstrusus in R. decollata snails and to investigate its potential as an intermediate host. For this purpose, first-stage larvae (L1) of A. abstrusus were obtained from the faeces of naturally infected cats. Doses of 500 L1/snail were given to 24 R. decollata snails, placed on the soil of the breeder chamber, and maintained under laboratory conditions. Three snails were killed at 8, 10, 12, 16, 22, 26, 45 and 55 days post-infection (dpi), and the muscular foot and visceral body were separately digested by an artificial digestion technique. The morphometric parameters of different larval stages were recorded. The mean number of larvae reaching the infective stage at the end of the study (L3) was 262 larvae/snail. The greatest development to L3 was recorded from days16 to 55 pi, during which the isolation was maximum. A. Abstrusus L3 were isolated from the viscera, but isolation from the snail foot was significantly higher. Our results showed for the first time the ability of A. Abstrusus larvae to develop in R. decollata, thus serving as a potential intermediate host.

The first report of Aelurostrongylus falciformis (Schlegel, 1933) (Nematoda, Metastrongyloidea) in badger (Meles meles) in Poland

Aelurostrongylus falciformis belongs to the superfamily of Metastrongyloidea. This nematode occurs in European badgers and locates in lungs, in respiratory tract. Numerous species of land snails are intermediate hosts of the parasite. In 2015, parasitological necropsy of 9 badgers, shot in the Forest District Gleboki Bród in Augustowska Primeval Forest, was performed. Two examined animals were infected with nematodes A. falciformis. In the lungs of each badger two specimens of nematodes were detected (male and female). In the following article, description, morphometrical data and figures are presented. This is the first report of A. falciformis infection in badgers in Poland.

Eosinophilic encephalomyelitis in horses caused by protostrongylid parasites.

Four thoroughbred horses showing lameness, ataxia, circling, depression, recumbency, and seizures, were examined. The horses had gross, pale- to dark-red manifestations and foci in the central nervous system (CNS). Multifocal to coalescing eosinophilic necrotizing encephalomyelitis was observed histologically in the CNS along with intact or degenerated nematodes. Nematodes had polymyarian-coelomyarian musculature, a smooth thin cuticle, and intestines lined by multinucleated cells with microvilli. These traits suggested the nematodes belonged to the family Protostrongylidae, which includes Parelaphostrongylus tenuis. It was concluded that the horses were infected by nematodes, presumably Parelaphostrongylus tenuis, resulting in eosinophilic necrotizing encephalomyelitis.

Metastrongyloid infection by Aelurostrongylus abstrusus, Troglostrongylus brevior and Angiostrongylus chabaudi in a domestic cat.

The "cat lungworm", Aelurostrongylus abstrusus, affects cats worldwide. Recently, other cardio-pulmonary parasites, e.g. Troglostrongylus brevior and Angiostrongylus chabaudi, have been isolated from cats either for the first time or a long time after they were first described. This paper describes the first known mixed infection by A. abstrusus, T. brevior and A. chabaudi in a domestic cat. Biological and epidemiological implications of old and "new" metastrongyloid-caused infections in cats are discussed.

Simultaneous infection by four feline lungworm species and implications for the diagnosis.

Besides Aelurostrongylus abstrusus, other parasites belonging to the superfamily Metastrongyloidea, namely Oslerus rostratus, Troglostrongylus brevior and to the family Trichuridae, i.e. Eucoleus aerophilus (syn. Capillaria aerophila), have also been reported as agents of respiratory infection in domestic cats. A case of simultaneous infection by four feline lungworm species in Sardinia is herein described. An adult female cat (Felis silvestris catus), road-killed in the southeast part of Sardinia (municipality of Villacidro, province of Cagliari), Italy, was referred to the Laboratory of Parasitology of the Veterinary Teaching Hospital in Sassari. At necropsy, the lungs were examined and dissected under a stereomicroscope for the presence of parasites, and first-stage larvae (L1) of broncho-pulmonary nematodes were searched for in a faecal sample using the Baermann method. Parasites collected in the lungs were morphologically identified as A. abstrusus, E. aerophilus, and O. rostratus. In addition to the above species, L1s of Troglostrongylus spp. were detected at coproscopy but no adult specimen was found in the lungs. The morphological identification was confirmed by the molecular amplification and sequencing of cox1 mitochondrial gene, 18S and ITS2 ribosomal DNA. This finding stands as the first simultaneous infection by four feline lungworm species in the same animal, and as the first report of O. rostratus and E. aerophilus in Sardinia.

Resurrection and redescription of Varestrongylus alces (Nematoda: Protostrongylidae), a lungworm of the Eurasian moose (Alces alces), with report on associated pathology.

BACKGROUND: Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series. METHODS: Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids. RESULTS: Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography. CONCLUSIONS: Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation.

First report of Troglostrongylus brevior in a kitten in Greece.

The first case of a natural infestation with Troglostrongylus brevior in a kitten in Greece is described here. A ∼40-day-old stray cat was referred to a private veterinary clinic with signs of respiratory distress. First stage larvae of a metastrongyloid nematode were observed in the wet mount faecal preparation. Despite an anthelmintic treatment, the respiratory signs worsened and the kitten died 2 days later. The larvae in the faeces were identified morphologically and genetically as T. brevior. The present evidence suggests a vertical or direct infestation of the kitten and a severe pathogenic role of T. brevior in young cats. This report expands the recent published cases of troglostrongylosis in domestic cats to a wider geographical distribution and opens new questions on the apparent spreading of T. brevior from wild to domestic hosts.

Feline lungworm Oslerus rostratus (Strongylida: Filaridae) in Italy: first case report and histopathological findings.

Oslerus rostratus syn. Anafilaroides rostratus (Strongylida: Filaroididae) is a metastrongyloid transmitted by snails, which localizes in peri-bronchial tissues and in the lung parenchyma of wild as well as domestic cats. In Europe, this nematode has been reported only on two occasions, being diagnosed in cats from Majorca Island and in northern Spain. Here, we describe a case of O. rostratus infection in a necropsied 4-year-old cat in Sicily (southern Italy). At the inspection of lungs, slender and greyish nematodes (four females and two males) were found embedded in the peri-bronchial tissues and in the bronchial walls. Parasites were morphological and molecularly identified as O. rostratus, with their 18S sequences being identical among them and showing a high homology (99%) with those available in public databases. At the histology, nematodes were encapsulated in a pseudo-cystic formation surrounded by an interstitial inflammatory process and fibrous tissue. Lung lesions were mainly represented by peri-luminal fibrosis, hyperplasia and hypertrophy of the bronchial mucosa and glands, respectively. This first record of O. rostratus infection from Italy indicates that this parasite should be included in the differential diagnosis of feline of lungworm infection.

Pathological and histological findings associated with the feline lungworm Troglostrongylus brevior.

Troglostrongylus brevior is a neglected feline lungworm species, which has been increasingly reported in the Mediterranean area, although scant data are available on the respiratory alterations it causes in cats. Therefore, we describe the gross and histological lesions of a 20-week old kitten that succumbed due to the onset of a fulminant respiratory failure. At necropsy, a catarrhal exudate was observed in the airways, along with nematodes in the trachea and bronchi. The lungs were processed for histological examination and serial pulmonary sections were performed. A total of 14 nematodes were collected, being all morphologically and molecularly identified as T. brevior. Lungworms were histologically localized within the bronchial lumen, surrounded by an eosinophilic infiltrate. The presence of T. brevior in the airways has been histologically documented for the first time and its life-threatening potential is discussed.

New insights on metastrongyloid lungworms infecting cats of Sardinia, Italy.

In addition to the well-known Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been diagnosed as a causative agent of bronco-pulmonary infections of cats in Spain and Italy. However, information concerning the impact of this species of lungworm on feline population is limited to a few case reports. From July 2011 to May 2013 an epidemiological survey was carried out on Sardinia island (Italy), where 107 individual faecal samples were examined by Baermann technique, and first-stage larvae were identified based on their morphology and characterization of molecular markers. The 29.9% (32/107) of cats examined were infested by broncho-pulmonary nematodes and, although A. abstrusus was the most frequently detected (n=27; 25.2%), larvae of T. brevior were also found (n=7; 6.5%). In addition, two cats (1.9%) were co-infested by both species. Overall metastrongyloid infection was higher in female cats (n=22; 38.6%) than in males (n=10; 20%) (χ(2)=4.39; p=0.036). The mean age of positive animals was 21.1 (±29.8) months, being infected animals from 2 months to 10 years of age. Of the 32 animals that scored positive for lungworms only 6 (18.8%) displayed a respiratory condition associated with lungworm infestations. Biomolecular characterization confirmed the morphological diagnosis of A. abstrusus. Positive samples that were identified at genus level as Troglostrongylus spp. were molecularly characterized as T. brevior. This study represents the first epidemiological survey on metastrongyloid lungworms of domestic cats in Sardinia and the first report of T. brevior on this island.

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ≈ 4.0 to 4.0 × 10(-5) µg/µl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10(-3) µg/µl of DNA for A. abstrusus, 4.9 × 10(-3)µg/µl for T. brevior, and as low as 4.0 × 10(-3) µg/µl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

Mixed infection by Aelurostrongylus abstrusus and Troglostrongylus brevior in kittens from the same litter in Italy.

Parasitic nematodes affecting the respiratory system of felids are spreading in endemic regions and emerging in previously free areas and/or hosts. This is particularly the case of the cat lungworm Aelurostrongylus abstrusus, which can cause respiratory signs in cats all over the world. Additionally, Troglostrongylus brevior has been recently found in domestic cats from Ibiza Island in Spain and Southern Italy. The present paper describes the first mixed infection by these lungworms in kittens belonging to the same litter. Two ∼10-11 weeks old kittens were found infected by A. abstrusus and T. brevior at a copromicroscopical examination. The identity of larvae shed by faeces were confirmed with an already validated PCR specific for A. abstrusus and a novel DNA-based assay specific for T. brevior. One kitten showed severe respiratory signs and died despite an anthelmintic treatment, while the other had a subclinical infection and recovered after a parasiticide administration with milbemycin oxime. New insights into epidemiology, biology, clinical aspects and control of these parasitoses are discussed.

Infection with Crenosoma striatum lungworm in Long-eared Hedgehog (Hemiechinus auritus) in Kerman province southeast of Iran.

Hedgehogs are distributed in different areas of Iran. Unfortunately, clinical and parasitological studies on parasites of hedgehogs are very few. Crenosoma striatum is a common lungworm in hedgehogs. C. striatum infection can cause weight loss, dry cough, bronchitis with ulcerous reactions based on secondary bacterial infections, pulmonary damage, thickening of the tracheal wall, and pulmonary emphysema up to cardiovascular failure. In this survey, six dead hedgehogs (Hemiechinus auritus) were investigated for lungworm infection. All the six hedgehogs had C. striatum infection in their lungs.

Crenosoma brasiliense sp. n. (Nematoda: Metastrongyloidea) parasitic in lesser grison, Galictis cuja (Molina, 1782) (Carnivora, Mustelidae) from Brazil, with a key to species of Crenosoma Molin, 1861.

This study describes Crenosoma brasiliense (Nematoda, Metastrongyloidea), a new species parasitic in bronchi and bronchioles of Galictis cuja (Molina) (Carnivora, Mustelidae) from Brazil. This species differs from other 11 species of Crenosoma by having a cuticular projection at the distal end of the spicules, forming a prominent blade at the tip of the spicule, a vulval cuticular appendage with a triangular shape and prominent vulval lips. There are no previous records of species of Metastrongyloidea in G. cuja or species of Crenosoma in South America. Therefore, the new species represents the first host record and first geographical record of species of Crenosoma in South America.

Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) as agents of broncho-pulmonary infestation in domestic cats.

BACKGROUND: Aelurostrongylus abstrusus is currently regarded as the main metastrongyloid infesting domestic cats, whereas the reports of Troglostrongylus spp. in domestic and wild felids largely remain anecdotic. This paper reports on pulmonary infestation caused by Troglostrongylus brevior and Troglostrongylus subcrenatus in two kittens and describes, for the first time, associated clinical presentations and pathological features. Morphometrical, molecular and phylogenetic analyses have also been conducted to differentiate here the examined Troglostrongylus species from A. abstrusus, towards a clearer delineation of metastrongyloids affecting cats. METHODS: Two kittens were referred for respiratory distress and hospitalized with a diagnosis of severe aelurostrongylosis, based on the presence of metastrongyloid larvae in the faeces. Despite prompt treatment, kittens died within 48 hours. Both kittens were submitted to necropsy to determine the cause of death. RESULTS: At necropsy, nematode specimens were found in the trachea, bronchi and bronchioles and were associated with respiratory signs (i.e., dyspnoea, polypnea, severe coughing and nasal discharge). Morphology and measurements of adult parasites found allowed the unequivocal identification of T. brevior and T. subcrenatus, even if first stage larvae were rather similar to those of A. abstrusus. Briefly, T. brevior and T. subcrenatus larvae were shorter in length and lacking the typical knob-like terminal end of A. abstrusus. Molecular and phylogenetic analyses corroborated morphological identification and provided data on mitochondrial and ribosomal DNA genes of T. brevior. CONCLUSIONS: Data presented here indicate that T. brevior and T. subcrenatus may cause major respiratory distress in domestic cats. Consequently, these two species should be included, along with A. abstrusus, in the differential diagnosis of cat bronchopulmonary affections and treatment protocols need to be evaluated. Through research on the biology, epidemiology and control of Troglostrongylus spp. infestations in domestic cats are advisable to implement current knowledge on these neglected metastrongyloids.