Pathological findings of Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) from Central Italy,with the first report of a disseminated infection in this host species

In Europe, the red fox (Vulpes vulpes) is considered the reservoir of Angiostrongylus vasorum, nematode residing in the pulmonary arteries and right heart of dogs and many species of wild carnivores. Italy is considered one of the European countries where this nematode is actually spreading. Between May 2007 and November 2013, 62 foxes found dead in Central Italy were necropsied. Right heart and pulmonary arteries were opened and checked for the presence of adult parasites. Impression smears from sectioned lungs were examined for the presence of first-stage larvae, and samples of lungs were processed for histological examination. In order to detect eventual disseminated infections, samples of heart, pulmonary lymph nodes, liver, kidneys, and brain of foxes positive for A. vasorum at necropsy or lungs histological examination were processed for histological examination. An overall prevalence of 43.5 % was recorded. Light, mild, and severe lung lesions were detected in 33.3, 22.2, and 25.9 % of infected animals, respectively. Severe lesions were more frequent in animals younger than 12 months. In five infected foxes (18.5 %), no gross lesions were observed, while for three animals, angiostrongylosis was considered the cause of death. A case of disseminated angiostrongylosis was detected and another one was suspected. This is the firs report of disseminated angiostrongylosis in the fox.     

Molecular identification of novel intermediate host species of Angiostrongylus vasorum in Greater London

Angiostrongylus vasorum is a parasitic nematode that can cause serious and potentially fatal disease in dogs and other canids. The aim of this study was to determine the intermediate slug species infected in nature by sampling sites in Greater London and Hertfordshire located within a known hyperendemic region. Overall, A. vasorum larvae were recovered from 6/381 slugs (1.6 %) by tissue digestion, and their identity was confirmed by PCR. Infected slugs originated from three different sites in the Greater London area: one in Waltham Forest and two in Bromley. Slugs parasitised by A. vasorum were identified by a combination of external morphological characteristics and molecular techniques and belonged to three different families: the Arionidae, the Milacidae and the Limacidae. This includes two new host records for the parasite: Arion distinctus and Tandonia sowerbyi. This is the first record of A. vasorum in the family Milacidae, indicating that the parasite has a broader intermediate host range than previously recognised.     

Infections with cardiopulmonary and intestinal helminths and sarcoptic mange in red foxes from two different localities in Denmark

Monitoring parasitic infections in the red fox is essential for obtaining baseline knowledge on the spread of diseases of veterinary and medical importance. In this study, screening for cardiopulmonary and intestinal helminths and sarcoptic mange (Sarcoptes scabiei) was done on 118 foxes originating from two distinct localities in Denmark, (Copenhagen) greater area and southern Jutland. Fifteen parasite species were recorded in 116 foxes (98.3%), nine parasitic species are of zoonotic potential. Parasite diversity was greater in foxes of Copenhagen in terms of overall parasite species richness and species richness of all helminth groups individually: trematodes; cestodes; and nematodes. Six parasite species were recovered from foxes of Copenhagen, but not from foxes of Southern Jutland: Echinochasmus perfoliatus; Echinostoma sp.; Pseudamphistomum truncatum; Dipylidium caninum; Angiostrongylus vasorum; and Sarcoptes scabiei, but Toxascaris leonina was only recorded in foxes of southern Jutland. A high prevalence and abundance of A. vasorum in foxes of Copenhagen was observed. The prevalence of four nematode species; Eucoleus (Capillaria) aerophilus, Uncinaria stenocephala, Toxocara canis, and Crenosoma vulpis, in foxes of both localities were comparable and ranging from 22.9% to 89%. The prevalence of Mesocestoides sp. was significantly higher in foxes of Copenhagen. Taenia spp. were detected using morphological and molecular analysis, which revealed the dominance of T. polyacantha in foxes of both localities. Infections with sarcoptic mange were evident only among foxes of Copenhagen (44.9%), which significantly affected the average weight of the infected animals. Further remarks on the zoonotic and veterinary implications of the parasites recovered are given.     

The common frog (Rana temporaria) as a potential paratenic and intermediate host forAngiostrongylus vasorum

Common frogs (Rana temporaria) were exposed either to third-stage larvae (L3) or to first-stage larvae (L1) ofAngiostrongylus vasorum. Following exposure to L3, viable larvae could be detected in the frogs for at least 2 weeks. Following exposure to L1, the frogs developed viable L3 in their tissues within 30 days. In a test of the infectivity of these larvae, one fox was fed frogs previously infected with L3 and another fox was fed frogs previously infected with L1. On autopsy it was found that adultA. vasorum populations had been established in both foxes. Thus, it could be concluded that frogs can act not only as paratenic hosts but also as intermediate hosts forA. vasorum.     

PCR detection of Angiostrongylus vasorum in faecal samples of dogs and foxes

The cardiovascular nematode Angiostrongylus vasorum is spreading in the fox and dog populations of northern Europe. A. vasorum can result in severe clinical manifestations in dogs; therefore, specific diagnosis is crucial for assessing its prevalence. In the present study, faecal samples from foxes and domestic dogs were tested by a new polymerase chain reaction (PCR) targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. vasorum. Initial isolation of faecal larvae by sieving facilitated the processing of larger sample volumes and allowed for the recovery of dead larvae from frozen samples. The sieve-PCR method enabled the identification of a single larva per 2 g of faecal sample and did not amplify DNA of a range of canine helminths, thus presenting a non-invasive tool for wildlife surveillance and for confirmative diagnosis in dogs.     

The first clinically manifested case of angiostrongylosis in a dog in Slovakia

The first clinical case of canine angiostrongylosis from Slovakia, previously infection-free country, is described. 18-month old male Bernese mountain dog living in south-eastern part of Slovakia showed poor health condition characterized by weight loss, irritating cough, dispnoe, intense salivation, vomiting and bilateral scleral bleeding. Two times even the acute physical collapse occurred. Blood analysis was provided and revealed increase of total protein, eosinophilia, monocytosis, and mild thrombocytopenia. Anaemia characterized by reduced number of erythrocytes and reduced levels of haemoglobin, packed cell volume and iron was also diagnosed. Larvoscopic Baermann technique revealed the presence of Angiostrongylus first stage larvae. Infected dog excreted larvae in high numbers — in 10 g of the faecal material more than 800 larvae were counted. DNA analysis using PCR confirmed the presence of Angiostrongylus vasorum species. The first clinical case of angiostrongylosis has evidenced that the new life-threatening parasitic disease of dogs has spread to the territory of Slovakia. A serious effort is therefore inevitable to increase the professional awareness and knowledge on diagnosis, treatment and prevention.     

The reindeer abomasal nematode (Ostertagia gruehneri) is naturally transmitted to sheep when sharing pastures

The increasing number of sheep (Ovis aries) in northern Finland, often alternately corralled with winter-fed reindeer (Rangifer tarandus tarandus), creates potential for cross-infection of gastrointestinal nematodes. The aim of this study was to elucidate this possibility with 43 animals. Eleven reindeer and 8 sheep had shared a corral by turns, reindeer during winters, and sheep in summers. Another 12 reindeer had no known contact with sheep. Twelve sheep had no close contact to other ruminants. Both reindeer groups were free-ranging during summers. During slaughter in September to November, 2003, abomasa and parts of intestines were collected. Gastrointestinal nematodes were counted and identified. The species found were the following: in reindeer, Ostertagia gruehneri/Ostertagia arctica, Mazamastrongylus dagestanica, Nematodirus tarandi, Nematodirella longissimespiculata and Bunostomum trigonocephalum; in sheep, Teladorsagia circumcincta/Teladorsagia trifurcata, O. gruehneri/O. arctica, Nematodirus filicollis and N. spathiger. In the sheep sharing corral with reindeer, the only abomasal nematode species found was O. gruehneri, a reindeer parasite. The generation interval of O. gruehneri in Finnish reindeer appears to be shorter than in Canadian Arctic caribou, where complete larval inhibition leading to only one generation yearly has been reported.     

New insights onto cardiopulmonary nematodes of dogs in Sardinia,Italy

Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4 % (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9 % (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.     

First autochthonous case of canine Angiostrongylus vasorum in Slovakia

Angiostrongylus vasorum is a metastrongyloid nematode that may cause cardiopulmonary disease, neurological signs and coagulopathies in dogs. The parasite has an indirect life cycle with molluscs as intermediate hosts, in which the infective third larval stage develops. Recently, A. vasorum has been repeatedly reported in dogs outside the endemic areas, indicating that this parasite is widely distributed over Europe. This is the first record of an autochthonous infection in a dog from Slovakia that was casually diagnosed during routine preventive parasitological examination. A. vasorum first-stage larvae were recovered using the Baermann technique and identified by length and characteristic tail morphology. The animal originated from Slovakia and had not travelled abroad. The dog had been regularly walked on grass fields with a concentrated presence of common species of Gastropoda and frogs. The owner reported that the dog had been licking and eating grass and it had shown curiosity for molluscs and frogs. The first finding of A. vasorum-infected dog in Slovakia has confirmed that the parasite is spreading beyond the traditional hyperendemic foci, which accentuates the need for monitoring and increasing of disease awareness in primary care clinical practice.     

Red fox (Vulpes vulpes) cannibalistic behaviour and the prevalence of Trichinella britovi in NW Italian Alps

Food habits of the red fox (Vulpes vulpes) were studied in the Aosta Valley region (NW Italian Alps) and were related to the prevalence of Trichinella infection in the red fox itself and in two Mustelid species (the stone marten (Martes foina) and the badger (Meles meles)). The search of Trichinella by the automatic digestion of muscles samples led us to determine a prevalence of 3.5±1.2% in red foxes, 7.9±4.3% in stone martens and 1.9±1.8% in badgers, with no significant differences among the species. All larvae were identified as Trichinella britovi. The fox diet was assessed through the analysis of both faeces (n=180) and the stomach contents of road-killed animals (n=109). Our results confirmed the opportunistic feeding behaviour of the red fox, which is able to use various trophic resources, both of animal and vegetal origin: e.g. wild and cultivated fruits (F%=47.1; V%=67.3), rodents (F%=22.8; V%=64.8) and carrion (F%=15.6; V%=78.6) formed the bulk of the foxs diet. The frequency of occurrence of potential events of cannibalism was 1.0%, even if the complete absence of undigested remains, other than hairs, suggested the possibility of confusing cannibalism with coat-cleaning. We suggest that intra-specific necrophagy could not represent the unique way of transmission of the nematode in natural conditions.     

Enolase of Angiostrongylus cantonensis: more likely a structural component?

The cloned enolase gene of Angiostrongylus cantonensis (AcEno) comprised 1,667 bp and encoded a peptide with 434 amino acid residues which lacked of a signal peptide but contained a transmembrane region, indicating that AcEno tends to be a structural component (intracellular or membrane protein). The real-time PCR revealed a meaningful difference in the expression level of AcEno in varied development stages. By immunolocalization, native AcEno was detected mainly in the cytoplasm in most tissues, such as parietal muscle, genital tracts, nerve ring, and alimentary canal where the energy consumption is high, but not as expected on the cuticle and hypodermis layer of the nematode. This suggests that the AcEno may be involved in a host of other biological functions, rather than a receptor of plasminogen or a component of excretory–secretory antigen. In addition, AcEno expressed alike in the nucleus, indicating that AcEno also involved in regulating the continuous growth and development of A. cantonensis in hosts. Despite of living in the vasculature at a certain stage of life cycle, AcEno was not localized in the outer surface of L3 and adults, indicating that A. cantonensis may have other virulence and immune evasion mechanisms.     

Pathology of naturally acquired Angiostrongylus vasorum infection in the red fox (Vulpes vulpes)

199 out of 509 foxes culled in a rabies control scheme, were shown to be infected with the metastrongylid parasite Angiostrongylus vasorum. Pathological lesions associated with angiostrongylosis in the fox are described and compared to reported lesions of A. vasorum infection in the dog. The lesions observed in the foxes seemed to be less severe and mainly confined to the ventral parts of the lung lobes which showed granulomatous pneumonia. Right ventricular hypertrophy of the heart was present in all infected foxes. It is believed that the fox population represents an important reservoir of A. vasorum.     

Description of Cloacina atthis sp. nov. from the stomach of the euro (Macropus robustus) (Marsupialia: Macropodidae) from Western Australia based on morphological and molecular criteria

A new species of strongyloid nematode from the genus Cloacina (Chabertiidae: Cloacininae) is described from the stomach of the hill kangaroo or euro (Macropus robustus) (Marsupialia: Macropodidae) from Western Australia. Cloacina atthis sp. nov. was found only in euros from the Pilbara region in the northwest of Western Australia, in spite of extensive collecting of the same host species from around the Australian continent. C. atthis is most closely related to Cloacina clymene, a species found in the same host species but only in the eastern half of the continent; the two species differ in minor morphological features (the shape of the wall of the buccal capsule, spicule lengths, the degree of sclerotisation of the gubernaculum and the shape of the vagina) as well as in differences in the internal transcribed spacers of ribosomal DNA. This study highlights the importance of using molecular methods when investigating the apparently disjunct distributions of strongyloid nematodes.     

Disseminated angiostrongylosis with massive cardiac and cerebral involvement in a dog from Italy

A case of disseminated angiostrongylosis caused by Angiostrongylus vasorum in a dog living in Italy is here described. The dog was referred for severe respiratory distress and epileptic seizures; clinicopathological findings were consistent with severe pneumonia associated with right-sided heart failure and multifocal involvement of the brain. Bronchoalveolar fluid analysis identified a multitude of nematode larvae, identified as A. vasorum by conventional and biomolecular (PCR) methods. The major anatomo-histopathological lesions were chronic granulomatous pneumonia, a severe multifocal granulomatous myocarditis and multifocal mild vascular and inflammatory disease in the brain. A. vasorum should be included among the differentials of dogs with cardiovascular and neurologic disease.     

The helminth fauna study of European common brown frog (Rana temporaria Linnaeus, 1758) in the Volga basin

In this paper we considered information on the helminth fauna of the European common brown frog (Rana temporaria Linnaeus, 1758) from 10 regions of the Volga basin. This study includes consolidated data of different authors over the last 30 years, supplemented by the results of our own research. There are reliably known finds of 29 species of helminths: Monogenea — 1, Trematoda — 21, Nematoda — 7. Trematodes Gorgodera asiatica Pigulevsky, 1945, Paralepoderma cloacicola (Luhe, 1909), mtc. and nematodes Icosiella neglecta (Diesing, 1851) were observed for the first time in a given host on the territory of Russia and the Volga Basin. Six species of worms make the basis of helminth fauna: nematodes Rhabdias bufonis, Oswaldocruzia filiformis, Neoxysomatium brevicaudatum and Cosmocerca ornata, trematode Haplometra cylindracea and monogenea Polystoma integerrimum. These six species are the most common and widespread parasites of the brown frog. For each species of helminths there is the following information included: taxonomic position, localization, area of detection, biology, definitive hosts, geographic distribution, the degree of host-specificity.     

Anisakids (Nematoda: Anisakidae) from stomachs of northern fur seals (Callorhinus ursinus) on St. Paul Island,Alaska: parasitological and pathological analysis

Anisakid nematodes parasitize the alimentary tracts of aquatic vertebrates, including northern fur seals (Callorhinus ursinus) (NFS). The main purpose of this study was to estimate the prevalence, intensity, and species composition of anisakids in NFSs on St. Paul Island, Alaska, and to analyze changes in NFS infection with these nematodes during the last decades. The study was carried out on St. Paul Island, Alaska, in July–August 2011–2013. Stomachs of 443 humanely harvested NFS males were collected from five separate haul-out areas. A total of 4,460 anisakid specimens were collected and identified by morphological criteria. Gross and histological examinations of stomachs were also performed. The overall prevalence of NFS infection was 91.2 %; overall mean intensity 10.9, and overall median intensity 6. Five species, Anisakis simplex s. l., Contracaecum osculatum s. l., Pseudoterranova decipiens s. l., P. azarazi and Phocascaris cystophorae, were found. The prevalence of Anisakis was 46.5 %, its intensity 7.5. The prevalence and intensity of Contracaecum, Pseudoterranova, and Phocascaris were 33.6 % and 3.5, 81.9 % and 6.5, and 5.2 % and 1.7, respectively. Significant differences in the prevalence of NFS infection were observed between separate haul-outs. Comparison of the present data with the results of studies performed in the North Pacific in the 1960s, showed a significant decrease in the intensity of NFS infection with anisakids during the last decades. The prevalence of mucosal lesions in NFS stomachs caused by anisakids also decreased from 92 to 20.9 %. Possible reasons for the changes in NFS infection with anisakids are discussed.     

Molecular identification of Heterakis spumosa obtained from brown rats (Rattus norvegicus) in Japan and its infectivity in experimental mice

Heterakis spumosa is a nematode of invasive rodents, mainly affiliated with Rattus spp. of Asian origin. Despite the ecological importance and cosmopolitan distribution, little information is available on the genetic characteristics and infectivity to experimental animals of this roundworm. Heterakis isolates obtained from naturally infected brown rats caught in 2007 in the city of Sagamihara, east central Honshu, Japan, and maintained by laboratory passages were subjected to mitochondrial sequence analysis and experimental infection in mice. Sequencing of the cox1 gene revealed that nucleotides of H. spumosa and previously examined Heterakis isolonche isolates from gallinaceous birds in Japan differed by 11.2–12.2 % that conforms to the range expected for interspecific differences. The two H. spumosa isolates differed by a single 138T/C non-synonymous substitution in the 393-bp mt sequence. In a dendrogram, the H. spumosa samples formed a subcluster with members of the nematode superfamily Heterakoidea, H. isolonche and Ascaridia galli. In an experimental infection study, ICR, AKR, B10.BR and C57BL/6 mice strains were inoculated with 200 H. spumosa eggs/head and necropsied at 14 and 90 days post-inoculation (DPI) when the number of worms was recorded. Eggs were initially detected in faeces from 32–35 DPI in ICR, AKR and B10.BR mice and the highest mean number of eggs per gram of faeces (EPG) was 4,800 at 38 DPI, 2,200 at 58 DPI and 800 at 44 and 72 DPI in ICR, AKR and B10.BR mice, respectively. No eggs were observed in faeces of the C57BL/6 mouse strain during the experiment. A similar number of juvenile worms were isolated from all mouse strains at 14 DPI, whereas no adult worms were detected in C57BL/6 mice at 90 DPI.     

First findings and prevalence of adult heartworms (Dirofilaria immitis) in wild carnivores from Serbia

Heartworm (Dirofilaria immitis) is a parasitic roundworm that causes a zoonotic disease known as dirofilariosis. Little is known about the role of wild carnivores serving as reservoirs in nature. Therefore, we examined 738 hearts and lungs of free ranging wild carnivores from Serbia to determine the presence of adult heartworms. During the period 2009–2013, the prevalence in golden jackals (Canis aureus) was 7.32 %, in red foxes (Vulpes vulpes) 1.55 %, in wolves (Canis lupus) 1.43 %, and in wild cats (Felis silvestris) 7.69 %. No adult heartworm specimens were found in beech martens (Martes foina), stone martens (Martes martes), European polecats (Mustela putorius), badgers (Meles meles) or otter (Lutra lutra). The highest recorded prevalence was in 2013 (7.30 %) and the lowest in 2012 (1.6 %). In jackals, the prevalence was higher in males (10 %) than in females (4.06 %), while in foxes the prevalence was 1.75 % in males and 1.26 % in females. The most infected host was a wolf in which 37 adult specimens were found. Because of the potentially significant role in the life cycle of D. immitis, populations of wild carnivores in Europe should be further examined and tested for heartworm infections.     

New species of Gorgoderina (Digenea; Gorgoderidae) and other helminths in Euphlyctis cyanophlyctis (Anura: Dicroglossidae) from Dehradun, (Uttarakhand), India

Gorgoderina spinosa sp. nov. (Digenea, Gorgoderidae) from the bladder of the water skipper, Euphlyctis cyanophlyctis (Anura, Dicroglossidae), from Dehradun, India is described and illustrated. Gorgoderina spinosa is the 6th Indian species assigned to the genus and is separated from its congeners based upon the morphology of vitelline glands and the presence of a spinose integument. Two additional digenean species, Diplodiscus amphichrus and Ganeo tigrinus, and 3 nematode species, Cosmocerca kalesari, Gendria chauhani, and unidentified larvae were found. Diplodiscus amphichrus, Ganeo tigrinus, Cosmocerca kalesari, and Gendria chauhani have previously been reported to infect Indian individuals of Euphlyctis cyanophlyctis.     

Comparative studies on the proteomic expression patterns in the third- and fifth-stage larvae of Angiostrongylus cantonensis

Angiostrongylus cantonensis is an important zoonotic parasite causing eosinophilic meningitis and eosinophilic meningoencephalitis in humans. In this study, the protein expression profiles of the infective third- and pathogenic fifth-stage larvae (L3 and L5) of this parasite were compared by proteomic techniques. Isolated protein samples were separated by two-dimensional gel electrophoresis (2-DE), stained with silver nitrate, and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Proteins from L5 were mainly at pH 5–7 and with molecular weight (MW) 40–100 kDa, whereas those from L3 were at pH 5–6 and with 5–35 kDa. Of 100 protein spots identified, 33 were from L3 whereas 67 from L5 and 63 had known identities, whereas 37 were hypothetical proteins. There were 15 spots of stress proteins, and HSP60 was the most frequently found heat stress proteins in L5. More binding and protein transport-related proteins were found in L5 including peptidylprolyl isomerase (cyclophilin)-like 2, serum albumin, preproalbumin precursor, and dilute class unconventional myosin. L3 had a higher expression of cytoskeleton and membrane proteins than L5. In addition, four protein spots were identified in the sera of the rat host by Western blot analysis. The present proteomic study revealed different protein expression profiles in L3 and L5 of A. cantonensis. These changes may reflect the development of L3 from the poikilothermic snails to L5 in the homoeothemic rats. This information may be useful for the finding of stage-specific proteins and biomarker for diagnosis of angiostrongyliasis.