Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

In Europe domestic and sylvatic canines and felines are the reservoirs of Dirofilaria immitis and D. repens , while different culicid mosquito species act as vectors of these species. Many mosquito species feed indiscriminately on animal reservoirs and man, thus where there is canine dirofilariosis, the risk of zoonotic infections exists. There are three forms of human dirofilariosis: Pulmonary dirofilariosis (PD), usually causing a solitary pulmonary nodule attributed to D. immitis ; sub-cutaneous dirofilariosis (SD) manifesting as subcutaneous nodules located in different parts of the body and ocular dirofilariosis (OD) in which worms cause nodules or remain unencapsulated in the eye area, being the last two variants mainly caused by D. repens . Most of the information on human dirofilariosis is generated by the clinical cases reported and their retrospective review, but there is very scarce other kind of studies. In Europe continues the sharp increase of SD/ OD cases unlike the extremely low number of reports of PD cases, without being able to indicate the objective causes of this fact,

As many as 3,545 cases of human dirofilariasis were recognized in Russia, Ukraine, and Belorussia starting from 1997.Clinical problems of human dirofilariasis become an issue and need be thoroughly analyzed.A patient self-assessment, the parasite anatomical location and clinical manifestation determine diagnostic workup.Five patients with peritonitis were operated immediately and Dirofilaria was unexpectedly found in peritoneal cavity.In contrastfive patients with "silent" pulmonary dirofilariasis were diagnosed late and accidentally.Affected eye (37% of all patients, variations 22 -48% in different years) in the case of a foreign "moving entity" in an eye or eyelid conjunctiva (19%) or with eye acute inflammation (25%) strongly motivated a patient to visit a doctor in the contrast with patients with slowly growing "silent" nodule (56%).Anyway as many as 86% of the patients with eye located Dirofilaria were addressed to a doctor during the first month of the disease.Nearly equal proportion of patients (around 62%) with head (28%), or trunk (12%), or man's genitalia (3%) located parasite also visited a doctor in the first month of the disease.Female patients with breast location (3%) were consulted earlier and were undergone surgery in short time (in the first 2 weeks) mostly because the main diagnostic hypothesis was breast cancer.In the cases of extremities located parasitic nodule (hands -9.4%, legs -8.6%)only 31% and 36% of patients (accordingly) decided to be consulted by a doctor in the first month from the onset because they did not regard their condition as life-threatening.The next important issue is doctor information about dirofilariasis, his specialty and previous experience.Everything had great influence on timely and correct diagnosis or at least on inclusion of dirofilariasis in the list of diagnostic hypotheses.In the territories with sporadic morbidity only few doctors (7%) suspected dirofilariasis before surgerythey mostly diagnosed benign or malignant tumors (72%).There is a contrast with endemic territories where dirofilariasis was suspected by doctors in the much higher proportion of the patients (85%).Preliminary ultrasound and color Doppler examination of patients with dirofilariasis made great input in the diagnosis.The findings include hypoechoic encapsulated linear structures without internal blood vessels and sometimes (47%) with detectable movement of the parasite.Those findings allowed to exclude malignancies before surgery in all ultrasound examined patients.Medical community has to be better informed about dirofilariasis.Ultrasound should be a standard procedure in patients with subcutaneous nodules.

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A few thoughts about the recent epidemiological situation of dirofilariosis in Hungary with particular regard to quick spread and high prevalences in certain areas Éva Fok 1 , István Kucsera 2  Heart worm disease caused by Dirofilaria immitis is well known in Southern parts of Europe.In the past decade several studies have been performed on its diagnosis, treatment and prevention, but knowledge on vector competence of Central European mosquito species for D. immitis under local climate conditions is still scarce.The aim of this study was therefore to analyze the vector competence of three different mosquito species (Aedes vexans, Ae. geniculatus and Culex pipiens biotype molestus, endemic in Serbia and Switzerland) for D. immitis at constant and realistic fluctuating temperature regimes under laboratory conditions.Mosquitoes were kept in the laboratory at 24-27 °C, with 85% of humidity and fed with sugar solution.Six groups of female mosquitoes (30-52 individuals), were fed with blood containing D. immitis microfilariae (6,000 mf/ml) obtained from a naturally infected dog using artificial feeding methods (Hemotek, a blood sausage, cotton stick method).After inoculation, blood fed mosquitoes were incubated at constant or realistic fluctuating temperature of 27 °C (17.5-35 °C, with average 27 °C).Parameters determined were: feeding rate, infectious dose, mortality rate, infection rates and infectivity rates.Mosquitoes were dissected under a stereomicroscope on days 2, 4, 7, 10, 14 post inoculation (p.i.), to observe the developmental stages of the filariae.Additionally, PCR analysis was performed.The feeding rate ranged from 26% in Cx pipiens biotype molestus to 61-63% in Ae.vexans groups and to 74% in Ae.geniculatus.The observed infectious dose was 7-10 microfilaria in Ae.vexans.No live microfilariae could be observed in the other species in samples taken on day 1 p.i. Mortality rate was rather high, it was altogether, 78% during the 14 days of incubation period and ranged from 12.5% in Cx.pipiens biotype molestus fed with cotton stick to 100% in those fed with Hemotek.The highest percentage (73.9%) of mosquitoes died until day 4 p. i (feeding).In Ae.geniculatus and Ae.vexans groups, microfilariae were found until day 7 p.i., L1 from day 4-10p.i. and L2 were found only in Ae.vexans groups on day 10.In Cx.pipens biotype molestus no larval stages were found by microscopy.PCR analysis revealed the highest number of specimens positive for Dirofilaria in Ae.vexans,k e p ta tc o n s t a n t2 7°C .A l lo ft h eCx pipiens biotype molestus mosquitoes that died during the experiment were PCR positive.Female mosquitoes developing infectious L3 stages (infectivity rates), L3 were identified only in Ae. vexans at day 14 p.i.Total infection rates (microscopy and PCR) were 72% for Ae vexans kept at constant 27 °C, 56.6% for Ae vexans kept at 27 °C fluctuating, 70% for Ae.geniculatus and 37.5% for Cx pipiens biotype molestus fed with the Hemotek.To conclude, vector competence for D. immitis was shown for the flood water mosquito Ae. vexans both at constant and fluctuating 27 °C.The respective results were not yet conclusive for Cx pipiens biotype molestus and Ae.geniculatus and further studies will be necessary with these species.Currently, the only registered drug for adulticide therapy in dogs with heartworm disease (HWD; Dirofilaria immitis)i sm e l a r s o m i n e dihydrochloride (Immiticide®, Merial).The American Heartworm Society (AHS), based on results from several studies [1,2], currently recommends that, in cases where arsenical therapy is not possible or is contraindicated, a monthly heartworm preventive along with doxycycline for a 4-week period might be considered.There is no published data on the use of moxidectin in combination with doxycycline.Preliminary results of an on-going study* show that moxidectin, the only macrocyclic lactone (ML) registered as a microfilaricide, is also adulticidal when combined with doxycycline.Interestingly, a similar combination therapy has been shown to be highly effective against human body lice, an ectoparasite that has been shown to develop resistance to MLs and which also harbours bacterial endosymbionts [3].It is not yet known if the efficacy of antibiotics and MLs is due to pharmacokinetic or pharmacodynamic synergism.It has been shown that compounds including antibiotics can increase intracellular concentrations of MLs and that MLs can inhibit cell detoxification mechanisms, thus increasing intracellular concentrations of drugs, including antibiotics [4].A recent study has shown, however, that serum levels of doxycycline in dogs treated with the combination protocol were not statistically different compared to dogs treated with doxycycline alone [5].It would be of interest, and a research priority, to elucidate the nature of this synergy.This study was funded by University of Parma (65/OPBA/2015).
Cortisol, a steroid produced in the adrenal cortex, is a key hormone involved in the stress response and serum levels have often been used as a measure of stress.It has been demonstrated that prolonged stress, as indicated by cortisol levels, is associated with reduced survival, fecundity, and immunity [1].Studies have examined interactions between parasites and cortisol levels in some species, with discrepant results [2].The aims of this study were to evaluate the potentially stressful effects of the infection of Dirofilaria immitis in dogs by measuring the levels of serum cortisol before and after the adulticide treatment.Serums from 61 dogs positive to D.immitis antigens were included; all blood samples were further examined by the modified Knott test.The parasite burden was assessed by echocardiography in 51 of these dogs [3].Furthermore, 22 dogs were treated following the AHS protocol and additional blood samples were taken on days 60, 90 and 120.Serum cortisol was measured by EIA Method, validated for this species.There were 24 females and 37 males.Thirty were clientowned dogs and 31 lived in a local shelter; 41 were microfilaremic and 20 were amicrofilaremic; 26 were symptomatic and 35 were asymptomatic.When the parasite burden was asses (n = 51), 20 had high burden and 31 had low burden.The mean level of cortisol in heartworm infected dogs was 10.08 ± 8.16 ng/ml.There were not statistically significant differences between sex and microfilaremic status, but there were between symptomatic and asymptomatic dogs (p < 0.05).When parasite burden was evaluated, dogs with high burden had significantly greater levels of cortisol (p < 0.001).During the adulticide treatment, the levels of cortisol dropped gradually in each sampling, being the cortisol levels from day 120 within the reference ranges (2.31 ± 1.02 ng/ml).Shelter versus client-owned dogs had higher cortisol levels (p < 0.05).The results demonstrate presence of stress in dogs infected by D.immitis, especially in symptomatic dogs, and those with high parasite burden similar to a previous study [4].These results are similar to other studies which evaluated the effect of several parasites in animals and humans; while the different results found in other research may be caused by the small virulence of the parasites studied [2].On the other hand, as the parasites are being removed, the levels of cortisol gradually decreased.Although not the aim of the study, we could observe that dogs from shelter had higher levels of cortisol, consistent with previous studies [5].

Trial registration/ Consent to publish
The study was approved by the ethical committee of Veterinary Medicine Service of Las Palmas de Gran Canaria University (MV-2016/ 07) and was carried out in accordance with the current European legislation on animal protection.
The vector-borne zoonotic parasite Dirofilaria repens causes cutaneous dirofilariosis.In humans, it can manifest as skin nodules in several anatomical regions or subconjunctival infections.Present in the Mediterranean area, many parts of Asia and presumably also in Africa, D. repens is apparently expanding its distribution to previously non-endemic areas in the old world.In Hongkong, a new species, Candidatus Dirofilaria hongkongensis, has been reported to cause cutaneous and subconjunctival infections of humans.The objectives of this study were to compare mitochondrial genomes from these parasites and to obtain data suitable for population genetic studies.Complete mitochondrial genomes of four adult worms from Italy, Croatia and India were obtained by either PCR followed by Sanger sequencing or Illumina MiSeq.According to cytochrome oxidase I sequences, worms from Europe and India were identified as D. repens and C. D. hongkongensis, respectively.This is the first report of C. D. hongkongenis from the Indian subcontinent.The mitochondrial genomes of D. repens and C. D. hongkongensis are essentially organized like those of other spirurida encoding 2 rRNAs and 12 proteins but lacking the atp8 gene present in most animal mitochondrial genomes.An approximately 2.5 kb fragment was amplified from Dirofilaria positive canine blood samples or macrofilaria from Europe (n = 42), Thailand (n = 2) and Vietnam (n = 1) and analyzed together with the corresponding regions of the full-length genomes.In contrast to the very high similarity between the European and Vietnamese samples, samples from India (C.D. hongkongensis) and Thailand were only distantly related to the European D. repens samples.Notably, genetic differences between these three Asian samples were higher than those observed within D. repens.Phylogenetic analysis did not support the current taxonomy of the Onchocercidae but was in agreement with other recent molecular studies using multilocus analysis.D. repens and C. D. hongkongensis sequences clustered together and were most closely related to Dirofilaria immitis.
In conclusion, differences between Dirofilaria spp.were considerably high while D. repens was shown to be genetically quite homogenous.Analysis of mitochondrial sequences supports the hypothesis that C.D. hongkongensis represents a distinct species and suggests that samples from Thailand might represent another cryptic species or a genetically diverged C. D. hongkongensis population.Investigations on a larger geographic scale including representative numbers of samples from regions not analyzed so far as well as development of microsatellite markers for fine mapping would increase our understanding of the population genetics of D. repens.Dogs represent the main natural reservoir for numerous helminths, including some species of filaria that have microfilariaes circulating in the blood flow.Of these, the most known canine filarioid species are Dirofilaria immitis, D. repens, Acanthocheilonema reconditum and A. drancunculoides transmited by different vectors (i.e.mosquitoes, fleas, lice and ticks) [1].In Romania, stray dogs still remain an unsolved issue, despite the efforts made by the authorities in the attempt to gather and place them in shelters towards adoption.The aim of this study is the screening of the infestation with filarioids in new cases brought in two enroled dog shelters surrounding Bucharest, Romania.During November 2014 and October 2015, 282 stray dogs have been brought in two shelters near Bucharest and have been tested.Morphometric analysis of microfilariae canine blood from the enrolled dogs were made by Knott's modified test.For the detection of the D. immitis antigens it was used an in-clinic rapid assay test based on enzyme immunoassay technique (SNAP® 4Dx®, IDEXX Laboratories, Inc., Westbrook, ME, USA).The identification of the species was made by a histochemical technique to demonstrate acid phophatase activity patterns in the microfilariae, using a comercial kit test (Leucognost SP®, Merck, Darmstadt, Germany) in accordance with the manufacturer's instructions.Out of 282 enrolled dogs, 32.62% (n = 92) were positive for at least one filarial species.The modified Knott's test showed the presence of circulating microfilariae in 78 samples (27.66%), and the serum of 67 samples was positive for D. immitis antigens using an immunoenzymatic assay.As a result of the histochemical technique there were identified three species of microfilariae and the global prevalence was 23.76% (n = 67) D. immitis, 9.57% (n = 27) D. repens and 0.71% (n = 2) Acanthocheilonema spp.In addition, coinfection with D. immitis and D. repens was found in four samples.Morphometric evaluation showed the following measurements of the length and width of microfilariae: 298.27/5.9μmo f D. immitis, 358.81/7.2μmo fD.repens and 264/4.6 μmo fAcanthocheilonema spp.The results of this study highlight the presence of three species of filarioids from the dogs brought to the enrolled shelters with a high prevalence of D. immitis (23.75%).This raises public health concern that merits more consideration by both veterinarians and physicians.

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Investigations on Dirofilaria repens infection in Polish dogslooking for the objective features of the infection Artur Dobrzyński 2 , Maciej Klockiewicz 1 , Magdalena Wysmołek 1 , Michał Czopowicz 3 , Marta Parzeniecka-Jaworska 4 , Joanna Nowakowska 5 , Ewa Długosz The skin dirofilariosis caused by D. repens has been recognized as an emerging disease in Polish dogs.Since first cases were diagnosed almost 10 years ago, now the infection is considered as an increasing epidemiological problem.The extensity of infection in some areas in Poland was estimated over 12-36% [1,2] within the local dogs' populations.Veterinarians have found this infection as a real threat, so general aim of this research was to find objective features of the infection which could be used to establish the treatment algorithm for vets.The investigation was based on cases reported to Small Animal Clinic of the Warsaw Faculty of Veterinary Medicine as well as of those admitted to other veterinary clinics of Warsaw area.There were 428 dogs preselected (suspected for dirofilariosis) included to this research.Animals underwent physical examinations and blood tests (morphology and biochemistry).At the end of the study results of this preselected group were compared with results obtained from finally diagnosedinfected dogs.Microfilariae were found in 42.8% of examined dogs.Subsequently, PCR and ELISA tests were performed to confirm the infection in possibly infected ones.PCR with differential primers was performed to reveal parasite DNA in blood [3].ELISA tests were based on adult D. repens somatic antigens to detect specific IgG in infected dogs.PCR revealed the additional 8.8% infected dogs.PCR tests also confirmed that all individuals were infected with D. repens.Results of ELISA indicate that D. repens infection results in high specific IgG titers in more than 80% of infected dogs.ELISA allowed to diagnose over 1/3 additional infected individuals, which have been previously found negative (by blood smear).The blood morphology and biochemistry revealed statistically significant erythropenia, limphopenia, thrombocytopenia, reduced haematocrit, and increased levels of alkaline phosphatase and creatinine in infected dogs.Results suggest that infection is associated with general symptoms and problems of liver and kidneys.Additionally, the comparison between infected and not-infected groups showed that skin dirofilariosis was more often (2.6x) found in dogs which did not received any anti ecto-parasite treatment.The results were used to set up the treatment algorithm for practitioners who are not familiar with this newly emerged disease.It is allowed to suspect infection when similar blood results are obtained, and

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Awareness and strategies about canine heartworm (Dirofilaria immitis) infection in private practices in Greece: preliminary results of an ongoing questionnaire survey Anastasia Diakou Heartworm (HW) infection of dogs is highly prevalent in some areas of Greece [1], but information about the prevention and treatment strategies implemented in the clinical setting is limited.In order to evaluate the perception of veterinarians on the prevalence and their experience on diagnosis, treatment and prevention of HW, a questionnaire survey was designed.The questionnaire was distributed by e-mail to the veterinary practitioners registered to the two major Hellenic veterinary scientific societies.Twenty questions were included, investigating the frequency of HW in each practice and the routinely implemented strategies on diagnosis, treatment and prevention.Until today, 134 questionnaires have been completed; 51.5% from the Northern and Central Greece (NCG) and 48.5% from the rest of the country (RC, continental and insular).The percentage of veterinarians reporting that they see at least one HW case per month, trimester, semester and year was 22.3%, 18.5%, 16.1% and 16.9%, respectively, while no cases of HW was reported from 26.1% of the participants.The criteria for suggesting prevention included the geographical area where the animal lived (88.8%), its lifestyle (72%), breed (13%), and the owner's compliance (41.1%).Most of the veterinarians (61%) suggest prevention measures all year round, while some (35.5%)only during the warm season of the year.Regarding treatment, 50% of the veterinarians consider as first choice the protocol endorsed by the American Heartworm Society (AHS) and the European Scientific Counsel Companion Animal Parasites (ESCCAP), while 16% apply a "slow kill" protocol.For the prevention of pulmonary thromboembolism 84% of the veterinarians suggest strict activity restriction, 67% administer prednisolone while 28% use aspirin.There is a recorded difference of awareness of HW between the NCG and RC veterinarians that could be attributed to the indication of higher prevalence of HW in NCG.Indeed, only 10.3% of the NCG veterinarians report absence of HW in their area, while the corresponding percentage in RC is 40.9%.In NCG, 73.5% of the veterinarians suggest appropriate preventive measures (endorsed by the AHS and the ESC-CAP) while in RC only 42.4% suggest such measures.Moreover, 85.3% of NCG veterinarians advise HW prevention for all dogs, while the respective percentage in RC is only 12.1%.These results suggest that although the majority of small animal practitioners in Greece appear to generally comply with the updated guidelines on the prevention and treatment of HW, the geographical area-based perceived risk for HW substantially affects the preventive strategy implemented.

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Atypical case of subcutaneous filariosis in a cat: do we expect Dirofilaria immitis there?Simone Manzocchi Subcutaneous dirofilariosis is a well-known disease caused mainly by Dirofilaria repens and described in several mammalian species including human, dog and cat [1].Additionally, early developing stages of the heartworm, Dirofilaria immitis, are rarely reported in subcutaneous localization from humans and dogs.To our knowledge, evidence of this condition has not been described in the cat yet, even if the feline host can be affected either by the classic adult-related heartworm form or heartworm-associated respiratory disease (HARD) caused by immature stages.A 2 year-old, spayed male cat was presented for three subcutaneous nodules on the head and trunk.The cat lived in Northern Italy and was regularly vaccinated and treated monthly with an antiparasitic spot on formulation containing selamectin (Stronghold®, Pfizer).One of the three nodules was surguically excided and examined.Histology showed in the subcutis the presence of a nodular lesion characterized by a severe inflammatory infiltrated composed by macrophages, small lymphocytes, with fewer eosinophils and mast cells, supported by a proliferation of mature fibroblasts (fibrosis).Inflammatory cells were multifocally surrounding sections of parasites identified as nematodes.The parasites were characterized by a thick cuticle with a smooth external surface, prominent and large lateral chords and a polymyariancoelomyarian musculature.Microscopic features were compatible with D. immitis morphology.[2] After extraction from the paraffin block, DNA of the parasite was amplified with a PCR (ribosomal 5.8S-ITS2-28S region), the PCR product were purified, cloned and thereafter sequenced.A BLAST search revealed 97% identitiy to D. immitis isolate EU182331 and only 79% of identity the next related sequence of Dirofilaria genus (D. repens).The cat tested negative for D. immitis antigenemia and the two remaining nodules disappeared spontaneously in a few months.Identification of a filaroid nematode with smooth cuticles in the subcutaneous tissues can be challenging.All species of the genus Dirofilaria are characterized by cuticular ridges, except from D. immitis and D. lutrae [2], with the latter described so far only in USA in the North American river otter.The parasite in the present case most likely represents an immature stage of D. immitis which developed in the subcutis (L3-L4) and was successively entrapped in this localization.The immunity of the cat, which is not a suitable definitive host for D. immits, likely played a role in preventing migration of the immature stage to the pulmonary arteries.To author's knowledge this is the first reported case of subcutaneous localization of D. immits in a feline host.

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In field retrospective study of "slow-kill "treatment efficiency on heartworm positive dogs in general practises in Serbia Nenad Milojković 1 , Momčilo Aranđelović 2 , Ljubomir Ćurčin 3 1 Veterinary Clinic "Vet Centar", 11000 Belgrade, Serbia; 2 Veterinary Clinic "Vet Alfa", 11000 Belgrade, Serbia; 3 Veterinary Clinic "Intervet", 11000 Belgrade, Serbia Correspondence: Nenad Milojković (nenad.milojkovic66@gmail.com) Parasites & Vectors 2016, 10(Suppl 1):A13 Northern parts of Serbia are hyperendemic for Dirofilaria immitis.A lot of suburban and rural areas may have overall prevalences up to 50% in dogs, and treating them is of great concern for veterinarians in the field.Two limitations make adulticide treatment almost impossible: 1. Immiticide® is not available on the Serbian market and ordering it abroad is expensive for the majority of owners whose dogs are confirmed as Heartworm (HW) positive; 2. It is very difficult, especially in the countryside to do appropriate diagnostics (echocardiography) in order to estimate prognosis after melorsamine treatment.Therefore, the "slow kill" treatment is the only reliably choice for the majority of veterinarians in the field.We have gathered data about HW positive dogs from eleven general practises.The total number of antigen positive dogs was 258.Owners of only 105 dogs were interested to treat their pets.Seven of them have stoped visiting their veterinarians after one month, and 32 dogs with severe disease (respiratory distress, right sided heart failure, caval sindrome) died within the first 3 months after diagnostistics.A total number of 66 dogs, with mild and moderate disease, have continued with "slow-kill" treatment.Schedule for treatment was intermittent application of doxicyclin (10 mg/kg every third month) and prophylactic dose of ivermectin (10 mcg/kg every 15 days).Therapy was given until the first negative antigen test.During the first 3 months 9 (13.64%)patients became antigen negative, between 3 and 6 months 3 (4.54%), between 6 and 9 months 29 (43.94%) and 14 (21.24%) between 9 and 12 months; 11 (16.64%)dogs become negative after more than 12 months.All dogs from the last group did not visit veterinarians regularly and were not on continuous therapy.We also want to remark that a lot of dogs in the study were not tested each month.Most of them were retested twice annualy, due to financial capabilities of their owners.Data from the study shows frequent owners neglecting to do any further dignostics and treatment of their HW positive dogs in suburban and rural areas.Those dogs persist as a reservoir for the disease.This fact demands more active education of the owners.On the other hand, dogs of committed owners, with mild and moderate disease, which are going to "slow kill" treatment, have a good chance to be cured.The first autochthonous infection of Dirofilaria repens and Dirofilaria immitis in the Czech Republic were reported in 2006 [1] using several diagnostic methods for detections of these parasites.Since then, Dirofilaria infection was repeatedly reported in dogs [2] and, recently, D. repens wasdetectedalsoinmosquitoes [3]andinhumans [4].Thepresence of D. immitis in the Czech Republic was established only on detection of antigen using the commercially available test.In past 10 years, detection of D. immitis microfilariae, PCR detection or clinical case of canine dirofilariosis caused by D. immitis were not reported from the Czech Republic.The aim of presented survey was to confirm or exclude the autochthonous infection of D. immitis in dogs from the Czech Republic and to determine the extent of endemic distribution of D. repens within the Czech Republic.A total number of 392 blood samples from dogs were examined using the modified Knott test, IDEXX SNAP® 4Dx® test and PCR amplifying the fragment of COI gene of filarial nematodes [5].Only D. repens was detected by Knott test and /or by PCR with prevalence 6.4% (25/392).Six out of 25 positively diagnosed dogs had no travel history outside the Czech Republic, so the autochthonous infection was proven in 3.4% animals.Almost all positive dogs had originated from Southern Moravia region except a single one, which was from Zlín region, 100 km north of other positive localities.D. repens prevalence demonstrated in our sample set is lower than previously published (9-24%), however, distribution of positive animals corresponds well with published presence of D. repens positive mosquitoes and with occurrence of cases of autochthonous human dirofilariosis from the same region.Our study confirmed the endemic occurrence of D. repens in the region of Southern Moravia in the Czech Republic.Importantly, no D. immitis was detected.Based on these results, and considering total absence of published clinical cases, microfilariae or PCR detection of D. immitis in the Czech Republic, we strongly recommend not to consider the Czech Republic as currently endemic for this parasite.This study was supported by COST CZ LD14048; survey was organized in the framework of the EurNegVec COST Action TD1303.Canine dirofilariosis has rarely been diagnosed in Austria before 2008.All dogs had a history either originating from an endemic country or staying abroad for a certain time.Dogs were identified by accidental finding of microfilaria in blood or urine samples or by the directed detection of adult D. immitis or D. repens [1,2].From 2008 on case numbers increased rapidly regarding both infections.The typical origin from Mediterranean countries in dogs with heartworm disease has been replaced by the origin from Eastern countries, led by far by cases from Hungary (Fig. 1).Several animal welfare associations located in Austria financially support foreign animal shelters in neighbouring countries and organize dog importation to Austria and Germany on a large scale.Unfortunately, most of these animals are not tested for dirofilariosis prior to importation and they are not protected by microfilarizides to avoid local transmission to mosquitos.First canine cases of D. repens infections with probable autochthonous background have been diagnosed in Austria in 2008 [3].In 2014 first detection of D. repens in vector mosquitos has been reported [4] and confirmed by additional autochthonous canine cases.An obvious increase of imported dogs from eastern countries to Austria has been recognized within the last five years, concurrently canine heartworm disease cases increased markedly, too (Fig. 2).Several criteria turned out to be important in the consultation talk to the dog's owner for the decision to have these animals on therapy: 1) Several animal welfare association members refuse heartworm therapy in dogs due to possible side effects.Side effects and lethality rates were massively overstated and erroneously reported to the owners.2) Estimated costs are high, especially when following the guidelines from the American Heartworm Society 3) None of the owners was informed about the possible influence of importing infected dogs to Austria regarding endemicity and zoonotic hazards.
To offer a safe and affordable therapeutic regime, a modified scheme has been introduced to these animals including two injections of melarsomine three days apart, and oral medication of macrocyclic lactones and doxycyclin.Transmission risk was reduced immediately and 22/26 became negative in the antigen test within 4-8 months after melarsomine injections.Informing and advising animal welfare associations and dog owners as well as identification of imported and infected animals and the rapid onset of a safe therapy is the only way to delay or even avoid heartworm disease becoming endemic in Austria.
Cardiopulmonary dirofilariosis caused by Dirofilaria immitis, is a zoonotic mosquito-borne disease with a worldwide distribution and a potentially severe outcome in companion animals.Wild canids constitute a reservoir for many vector-borne diseases, by harbouring their vector-transmitted etiological agents [1,2].Red foxes (Vulpes vulpes) are the most abundant wild carnivores in Europe, their populations are increasing and they are invading many urban areas due to a good adaptation to human environments [1].Portugal is an endemic country for dirofilariosis in domestic dogs [3], although information on the occurrence of D. immitis in wild canids is sparse.An epidemiological survey was conducted to investigate the prevalence  One of the samples that were positive to D. immitis was obtained with meat juice, a finding which suggests that it could be used as an alternative sample to serum for the antigen detection of antigen, in post-mortem analysis.The present report demonstrates that infection with D. immitis is prevalent in red fox populations in Portugal, showing an increase of prevalence compared with recent reports [4,5] and suggesting a role of these animals as potential reservoir hosts for domestic pets and even to humans.Given the complex interaction between wildlife and domestic animals, humans and parasites, a robust health risk surveillance assessment should be implemented in Portuguese fox population to allow a better management of its vectorborne infections and diseases, in line with the 'One Health' concept.

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In search of Dirofilaria immitis in domestic dogs from Luanda Ana Margarida Alho 1 , Hugo Vilhena  [4].Despite the various pathogens previously described in this population [1,2], D. immitis antigen were not found in any of these dogs.D. immitis has already been reported in other sub-Saharan African countries, including Kenya [5], Mozambique [6] and Zambia [7], although to the best of the authors' knowledge there is no report of D. immitis in Angola.Nonetheless, we cannot exclude the existence of D. immitis or another related species (Dirofilaria repens,f o r example) that is not detectable with this routine test.Unfortunately, no blood was available to perform the modified Knott's technique to assess potential microfilaremia.Considering the zoonotic risk of this parasite and the presence of potential vectors, further studies are needed to characterize the current epidemiological scenario of filarial species in vertebrate hosts and vector insects in Angola.
Dirofilaria repens is not a life-threatening parasite, however is one of the major differential diagnosis that must be done when blood microfilariae are detected in a dog.This was the case in a 20 monthold Tosa dog that came to the surgery consultation of the Small Animals Veterinary Hospital of Alfort, France in February 2016.The dog had a subcutaneous skin nodule on the head.The medical imaging examination, performed the day of the consultation, showed a well-defined nodule of 1.5 cm of large and 5 mm of depth, with several hyperechoic lines inside.The nodule was punctured with a small needle and polynuclear granulocytes as well as microfilariae were detected, after staining.Blood analysis revealed the presence of microfilariae both in the smear and in the sediment following the Knott technique (mean 176 larvae/mL).After the surgical removal of the skin nodule, one nematode of 8 cm of length was found inside, identified by means of PCR (ITS) as Dirofilaria repens.Blood analysis performed 6 weeks later, in the absence of any treatment, demonstrated a decrease of microfilariemia of 22%.The dog was imported from South of Romania, at the age of 3 months, and since then, the dog never left Ile de France region.Since both in Romania and in Ile de France region, several cases of subcutaneous dirofilariosis have been described so far in dogs and cats, it is not possible to identify with certainty the place where the initial contamination occurred.However the region of south Romania, where the dog was born is highly endemic for mosquitoes.A stray queen with her offspring was delivered to the veterinary clinic in Warsaw.The litter consisted of 3 female and 4 male kittens.The age of kittens was estimated around 8 weeks.During physical examination all of them were found in poor condition.The family was severely infested with fleas.Fecal examination results showed that the queen was infected with Toxocara sp., Ancylostoma sp., and Dipyllidium sp., and the offspring with roundworms and hookworms.Some of the kittens manifested diarrhea and also conjunctivitis was noticed.A blood sample was collected from the adult cat to check its general status.During the examination some individual microfilariae were found.Blood samples were then taken from three kittens and blood smears revealed the presence of single microfilariae in two of them.Regarding the severity of the circumstances the veterinarian decided to apply moxidectin/imidacloprid topical solution (Advocate®, Bayer) and other necessary treatment.Two days later the veterinarian contacted our laboratory in the Division of Parasitology at the Faculty of Veterinary Medicine.We asked for blood and serum samples which were then taken from the queen and the kittens and delivered to our laboratory.In order to confirm the infection genomic DNA was isolated from blood samples and PCR was performed [1].PCR product specific for D. repens was amplified only in the sample originating from the queen which was taken before treatment.In queen and kitten blood samples which were taken after treatment PCR results were unambiguous.The presence of D. repens specific antibodies in all examined sera was confirmed by ELISA.The highest titer was noted in queen serum (1/25600).Titers in kitten sera were lower and ranged from 1/3200 to 1/800.Vertical transmission of filarial infections is uncommon.Only few cases of transplacental transmission of microfilariae have been reported: Brugia phanangi in the cat [2], Dirofilaria immitis in the dog [3], Wuchereria bancrofti and Onchocerca volvulus in humans [4,5].Our results allow to hypothesize that D. repens vertical transmission occurred in investigated cats.At the same time, it is very unlikely that kittens had been infected by another way at this age.In conclusion, there are many questions to be answered.What was the actual route of transmission in this particular case?What is the pattern of immune response against D. repens in cats?More research should be conducted in order to provide the adequate control measures to prevent skin dirofilariosis in pets and humans.Human dirofilariasis caused by a Dirofilaria repens is relatively rare zoonotic infestation, but according to the literature number of reported cases increase in Serbia, the Balkans and other European countries in the last 10 years [1,2,3].Recently, the parasite was identified by molecular techniques in Culex pipiens and Aedes vexans in Serbia [4].About 37 cases of superficial (subcutaneous and eye infections) and visceral infections were reported in Serbia till 2015 with predominant subcojnunctival and periocular infestation.Aim of this paper is to report new D. repens infections diagnosed in our country, and to address attention that this mostly benign infection may have serious clinical course.We present three cases of human dirofilariasis, two autochthonous and one imported, diagnosed from February 2015 till April 2016.All patients were adults, one male and two females, two with subcutaneous infection on the limbs and one with infection of the eye.The male patient, 57 years old is a resident of Belgrade who frequently travelled Novi Pazar which is located in the southern part of Serbia.He presented with five days history of pain, swelling and redness on the anterior part of the right thigh (6 ˣ 12 cm) near inguinal area, temperature 38 °C and eosinophilia (10.3).According to the clinical picture and ultrasound findings phlegmona and cellulites were diagnosed and ceftriaxone (2 g IV 5 days) was prescribed.Although pain and redness disappeared, and nodular swelling was clearly defined (5 cm), needle puncture was performed and white thread like 8 cm long mass was extracted and amoxicillin/clavulanic acid (100 mg twice/day for 15 days) was prescribed.Diagnosis of a D. repens infection was made in pathohistological preparations and the infectiologist prescribed ivermectin (200mcg/ kg PO once).He has been on follow-up for two months without any signs of recurrence.At the ocular case, a 64 year old woman from Belgrade was presented with history of progressive swelling, redness, pain and unpleasant feeling in the right eye conjunctival area.Under ophthalmic examination mass with thin, very active and movable worm wrapped in circles was found.The 8 cm worm was surgically removed under local anesthesia (Figs. 3 and 4).The third case of Dirofilaria infection was imported from Tivat, Montenegro in a 22 year old female.The infection was manifested as two weeks increasing subcutaneous nodule (3.5 cm) of the anterior forearm near elbow crease accompanying with pruritus, erythema and pain (Fig. 5).Routine blood tests, including eosinophil count were within normal limits.Abscess was diagnosed, incision was performed and 9 cm worm was extracted.

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In the previous two cases D. repens was identified according to morphological features in histological sections.All patients provided agreement for participation in this study.Several species of (family culicidae) mosquitoes have been identified as vectors of canine and feline cardiopulmonary dirofilariosis in different parts of the world.Its transmission depends mainly on weather conditions, which must be favorable for their development and survival.Europe is a continent where heartworm disease is expanding, but, at the same time, there are very few studies about the transmission vectors.Our aim is to review the current distribution of potential vectors in the European continent, the changes and their possible causes.Several factors can exert an influence on the emergence or discovery of species or new species to act as vectors, such as the climate change caused by the global warming or the interest in studying this disease in countries when new cases of canine heartworm are diagnosed.The activity of these species is another factor to consider.Mainly, their activity develops in spring and summer and their behavior depends of the different feeding patterns of each specie.For example, Cx. pipiens, Anopheles spp.are active only during the night while Ae.Albopictus predominantly at dawn or during the day.More studies and new programs of control of vectors in the current and new endemic countries, and control measures should be carried out to prevent the spreading of this disease.
Since dirofilariosis caused by Dirofilaria immitis is a vector-borne disease, its distribution depends on environmental conditions as well as demographic factors and the management of pets by humans [1].In the province of Salamanca (West-central Spain) the disease is known from many years, appearing in an area with extensive irrigated crops along the river Tormes [2].Because recent demographic changes have occurred in this area, the present study has been carried out with the aim to monitoring the distribution and prevalence of the disease in the canine population of this area.For that, 191 dogs were analysed through antigen and microfilariae tests and geo-referenced in a map.The general prevalence is 5.76%, although the disease is only present in dogs from municipalities with irrigated crops in which the prevalence is 16.67%.These results indicate that D. immitis continues to be present in the province of Salamanca and that it is associated with the presence of irrigations but with a clear decrease in the prevalence.Causes of the prevalence decrease as well as the potential zoonotic risk are discussed.

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Diagnostics and therapy of Dirofilaria immitis infections in an isolated dog shelter Vladan Panic 1 , Rastko Bekvalac Dirofilariosis is an important parasitic disease of dogs, cats and wild carnivores worldwide.It is among the most common canine vector-borne disease and represents a serious threat to both animal and public health [1].Greece is a typical Mediterranean country with reported cases of Dirofilaria-infected animals [2] and has favourable climatic conditions for mosquitoes, including new invasive species.The aim of this study was to investigate the presence of Dirofilaria spp. in clinically healthy dogs and to create a prevalence map including all geographic parts of the country.Blood samples were collected from a total of 276 dogs.They were animals of different breeds, both indoor and outdoor and used for different purposes (hunting, guarding, pets, shepherds, stray etc.).All samples were tested with the SNAP® 4Dx® Plus Test to detect Dirofilaria immitis antigen.In addition, samples were examined by Knott test to identify microfilariae of D. immitis and D. repens.Additional data were collected in order to identify potential risk factors.Thirty-two D. immitis antigen positive samples (11.6%) were detected by serology and19 of 32 were amicrofilariaemic.D. repens microfilariae were identified in 4 (1.4%) dogs.Infected dogs were originating significantly more from Northern than Southern parts of Greece (p < 0.01).Also, dogs at higher risk were the ones spending more time or activity outside the house (i.e.hunting) and with minimum preventive antiparasitic administration.These results revealed a high occurrence of Dirofilaria spp. in clinically healthy dogs in Greece and highlight the need to maintain a comprehensive and regular prophylaxis to reduce the contact between dogs and mosquito vectors.Furthermore, the findings of this study confirm that clinically healthy dogs need to be routinely screened for this parasite, as early diagnosis may be an important component of successful treatment and public health protection.
The study was funded by Bayer Animal Health GmbH.

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Subjective and objective assessment of radiographic findings in dogs with heartworm disease Ljubica Spasojevic Kosic Thoracic radiography is a very important diagnostic procedure for establishing a diagnosis of the heartworm disease (HWD).It enables an insight into the morphology of a lung field and cardiac silhouette.Radiographic changes associated with HWD can be assessed both subjectively and objectively.The aim of this work is to score subjective changes associated with canine heartworm disease in order to make them more comparable and useful for clinicians.Within objective measurements, in addition to the determination of a heart size, sizes of relevant blood vessels were determined according to the vertebral heart scale (VHS) system.Thoracic radiographs from 20 dogs with natural heartworm disease were measured.Both recumbent lateral (LL) and dorsoventral (DV) radiographs were available from 16 dogs whilst lateral recumbent radiographs were available from 4 dogs.The diagnosis of the heartworm infestation was established according to the results of wet blood smears, modified Knott test [1] and heartworm antigen test.Radiographs of each dog were assessed subjectively (vascular, alveolar and interstitial pattern and right-sided cardiomegaly) [2] and objectively (VHS) [3,4].A stage of heartworm disease was determined for each dog.Results were statistically analyzed and presented as percentages (qualitative variables) and mean ± standard deviation (SD) (quantitative variables).In this des c r i p t i v er e t r o s p e c t i v es t u d yw ed e f i n e da ni n c i d e n c eo fe a c h radiographic change and scored them, and calculated sizes of a heart and blood vessels relevant to the HWD among examined dogs.The most common radiographic changes subjectively assessed were increased sternal contact (95%) and rounding of the cranial border (90% of dogs).Scores for subjective assessment of radiographic findings in examined dogs were in the range 2/9 to 5/9 for cardiomegaly and 1/12 to 6/12 for lung pattern.Average heart sizes measured in LL and DV radiographs were 10.75 ± 0.78v and 11.04 ± 0.42v, respectively.Measurements of relevant blood vessels were as follows: vena cava caudalis 0.83 ± 0.10v, right cranial lobar artery 0.31 ± 0.08v and right caudal lobar artery 0.96 ± 0.42v.Further studies are needed to compare these results with results of dogs without HWD in order to define the most important changes that could be used as a diagnostic or prognostic tool.Objectivity in the assessment of the rad i o g r a p h so fd o g sw i t hH W Di sp o ssible to achieve by scoring the findings and using objective radiographic measurement.This work is part of the research done in the project TR31084 granted by the Serbian Ministry of Education and Science.

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Occurrence and taxonomical classification of microfilariae in blood samples from canine blood donors localized in south-eastern Poland Tomczuk Krzysztof 1 , Szczepaniak Klaudiusz  [2], which was confirmed in our study.Currently a cross-serological survey also revealed a circulating antigen of D. immitis in these geographical areas.In Poland 0.015% dogs were positive for circulating antibodies D. immitis [3].Despite the fact that dogs cannot infect Dirofilaria spp.via blood transfusion, the risk of spreading the reservoir invasion and possible immune reactions of the host (blood recipient) indicate that screening tests for dirofilariosis are essential.
In the past decades both Dirofilaria immitis and D. repens have spread from historically endemic areas to central and eastern European countries.Several studies have shown that Dirofilaria species are present in the southern and south-eastern areas of Romania [1].However, information about the vectors in the Danube Delta and their vector competence is lacking.In July 2015 more than 5,000 mosquitoes were collected in the Danube Delta in Romania at various locations (including mosquito traps next to a dog infected with both D. immits and D. repens).Mosquitoes were classified to specieslevel using the key after Becker et.al.[2].In one part of the study specified mosquitoes were pooled (up to 25 individuals per day/trap/ mosquito species).DNA was extracted and the samples were screened for filarioid helminths using conventional PCRs.Angiostrongylus vasorum, the French heartworm, is a parasite of dogs described in several parts of the world, including continental Europe and the British Isles [1].Regarding parasite occurrence in Scandinavia, endemic foci are widely present in Denmark and recently the parasite has been found in foxes in Norway [2].In Sweden the parasite was first identified in 2003 on the island of Sydkoster (Västra Götaland County) [3].Since then Swedish sporadic endemic cases of A. vasorum were diagnosed through positive canine faecal samples every year since 2011.A progressively increasing number of faecal samples has been submitted to SVA (National Veterinary Institute, Uppsala), however the prevalence in dogs appears to be quite low.A large-scale collection of canine serum samples was planned in order to identify the presence and distribution of A. vasorum in Sweden using more sensitive methods, i.e. serological methods able to detect parasite antigens and antibodies developed against the parasite.In this first large scale survey, 3886 sera from pet dogs were collected from the Clinical Chemistry Laboratory of the University Animal Hospital ( U D S -S L U ,U p p s a l a )a sw e l la sf r o mS V Aa n d3 3 0 9( 8 5 %o f3 8 8 6 ) have been tested until now by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA detecting specific antibodies against the parasite.Among the analysed samples a total of 0.39% (n = 13, 95% Confidence Intervals, CI: 0.21-0.67%) of the animals were positive in both ELISAs, while 0.70% (n = 12, CI: 0.44-1.04%) of the tested dogs were antigen-positive only and 1.48% (n = 49, CI: 1.48-1.10%)were positive for specific antibodies only.These preliminary results confirm that A. vasorum is established in Sweden with a prevalence comparable to other European countries.Definitive results from ongoing analyses will provide a deeper insight on the dissemination of the parasite over the country.Angiostrongylus vasorum and Crenosoma vulpis are important lungworms infecting dogs and wild canids, and their incidence is increasing worldwide.In Europe, red fox (Vuples vulpes) is considered as major reservoir host of these species.With regard to successful antirabies vaccination programmes and their urbanisation, red foxes represent significant infection risk for dogs.Despite it, data on the occurrence of these parasites in fox populations are very scant in a lot of European countries.In Slovakia, A. vasorum in dogs was for the first time reported in 2013 and then in 2014 [1,2].Consequential serological survey confirmed circulating A. vasorum antigen or the parasite-specific antibodies in 6.22% of dogs investigated [3]. A. vasorum was not previously reported in Slovak red foxes.On the other hand, C. vulpis was noticed in 1960ties and 1980ies in red foxes from Tatra National Park, Northern Slovakia, but its distribution and prevalence rate has never been formally surveyed.Therefore, the aim of the present study was to uncover real occurrence of A. vasorum and C. vulpis in fox population throughout Slovakia and estimate the risk of infection for dogs.Between September 2015 and April 2016 faecal samples of 420 red foxes were examined using flotation technique with zinc sulphate and Baermann migration method.The first stage larvae were determined by morphometric and morphological characteristics.Of 420 red 80 (19.05%) were positive for C. vulpis and 25 (5.95%) for A. vasorum.Only one fox showed dual infection with the both species.Geographic information system was used to map the spatial distribution of infected foxes.In conclusion, it should be said, it is the first monitoring of A. vasorum and C. vulpis in Slovak foxes and the data obtained will serve for any future epidemiological researches.

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Canine respiratory disease due to helminth infection is considered infrequent.Diagnosis is challenging due to poor detection sensitivity of fecal flotation for most species of lungworms.Along with an overreliance in clinical practice on fecal flotation for detection of parasitism, this leads to the potential for under-diagnosis of lungworms.A further complication is the sporadic fecal larval shedding patterns typical of metastrongyloid infections.Fecal samples (3 consecutive day collections) from dogs showing signs of respiratory disease were examined for the presence of lungworm first-stage larvae (L1) or eggs using the Baermann technique and zincsulfate centrifugal flotation from October 2014 to May 2016.Afrebrile dogs showing signs of respiratory disease (mainly chronic cough) that had not received an anthelmintic (excepting pyrantel or selamectin) within the last 60 days were included in the study.Baermann examinations were done on a 12-gram composite sample (4 grams of feces from each of the 3 collection days) and a 12-gram sample (day 3 collection) for each dog.Larval counts (L1/gram feces = LPG) were done on each of the 3 day collection samples if larvae were detected on either the composite or day 3 sample.Helminths known to cause respiratory disease were detected in 6 In recent years, infections with Angiostrongylus vasorum in dogs have increasingly been reported in European countries.For some time occurrence and distribution of A. vasorum seemed to be largely confined in isolated endemic foci.New reports of cases in dogs in endemic areas and data of post mortem surveys of foxes in areas previously believed to be free from infections suggest that A. vasorum has increased in prevalence and is spreading geographically within Europe.In Germany only few epidemiological studies have been performed and data on changes in the lungworm distribution in dogs in Germany are lacking.The aim of this study was to present actual data on occurrence and regional geographical distribution of A. vasorum and C. vulpis in dogs in Germany and to analyse these data in terms of evidence for geographically spreading of lungworms in Germany.In a retrospective study, the results of parasitological examinations of faecal samples, which had been submitted to the Veterinary Laboratory Freiburg, from 54,934 dogs between 2003 and 2015 in Germany were analysed.All faecal samples were obtained from privately owned dogs presented to local veterinary surgeons from all parts of Germany for mostly unknown clinical problems, routine examination and animal vaccination or general health check.All specimens were tested by a standardised flotation method with a saturated salt solution and examined by Baermann funnel technique to detect first-stage larvae (L1) of lungworms.The collected data were analysed by a geographic information system (GIS) using the programme RegioGraph 10 (GfK GeoMarketing, Bruchsal) to visualise the regional distribution of A. Angiostrongylus vasorum is a lungworm infecting dogs, foxes and few other wild carnivores [1][2][3].Reports of A. vasorum in dogs increased in the last two decades and foxes were frequently indicated as the relevant parasite reservoir, together with snails acting as intermediate hosts [4][5][6][7][8].
Our aim was to investigate the prevalence, worm burden and regional distribution of lungworms in Swiss red foxes, as well as to evaluate enzyme-linked immunosorbent assays (ELISA) for detection of circulating A. vasorum antigen and specific antibodies, which had previously been developed for dogs [9,10].Over the past five years lungs and hearts of 377 Swiss foxes were examined for the presence of A. vasorum and other lungworms.Blood collected from these foxes was used to evaluate the ELISAs.In the investigated fox population, A. vasorum, Capillaria aerophila and Crenosoma vulpis were identified: C. aerophila was found in all investigated cantons, whereas A. vasorum and C. vulpis did not occur in the canton of Graubünden.Overall prevalence of A. vasorum over the last five years was 45.1% (worm burden, WB: The adult stages of the nematode Angiostrongylus chabaudi (Strongylida, Angiostrongylidae), parasitize the pulmonary arteries and right ventricle of the heart and have been reported in wildcats (Felis silvestris silvestris) in Italy, in 1957 [1].Since that first description, A. chabaudi has never been reported, with the exception of the recent descriptions of immature stages in two cats in Italy [2,3].The case presented here is an infection by A. chabaudi in a wildcat from Northern Greece.The wildcat was found road-killed near the lake Kerkini (Macedonia, Greece).During necropsy, nematode parasites were found in the right ventricle of the heart and the pulmonary artery.The parasites were adult males and females and according their morphological characteristics were identified as A. chabaudi (Fig. 6).Additionally, parasitological examination of faeces and bronchoalveolar lavage revealed the presence of first stage larvae (L1) measuring 362-400 x 15-18.5 μm, with a kinked tail presenting a dorsal spine and a notch (Fig. 7).Both adults and larvae were subjected to molecular examination that confirmed that the parasites belong to the species A. chabaudi.The finding of histopathological examination of the lungs included heavy, extended, interstitial granulomatous pneumonia, with lesions detected around the larvae and eggs of the parasite.These findings were most likely, exclusively due to the presence of A. chabaudi, as there were no other parasites found in the lungs, suggesting that this parasite can be quite pathogenic to its hosts.The first description ever [4] of A. chabaudi L1 provides the necessary evidence that this nematode can complete its life cycle in the European wildcat, which should be considered its definitive host.The complete life cycle of the parasite remains unknown.For this reason, investigations that will include identification of intermediate hosts (most likely terrestrial molluscs), and development of the parasite both in the vertebrate and invertebrate host, are needed.The description of the diagnostic stage (L1) of A. chabaudi provides the basic information for future studies that will investigate infection in other feline species, e.g. the domestic cat and the implications to their health status.It is important to monitor in what extend can A. chabaudi affect domestic cats, a scenario that is possible but seems sporadic, according the recent available information of immature, unfertilized, not fully developed parasites isolated from domestic cats.In the last years lungworm infections of canids and felids have been the focus of special attention due to their emergence in several countries and spread into non-reported areas [1,2,3].Slugs and snails have been reported as intermediate hosts of these metastrongylids [4,5].Haemocytes, a denomination of cell types in invertebrates that freely circulate in the haemolymph [6], are involved in several physiological functions like coagulation and innate immune response [7].Haemocytes are similar to mammalian phagocytes, able to produce Extracellular Traps [8].This phenomenon, denominated ETosis, consists in an programmed cell death form in which the chromatin and antimicrobial proteins are expulsed into the extracellular region and finally induce the formation of fiber-like structures, that have the capacity to trap and inactivate pathogens of diverse kinds, like bacteria, viruses and others parasites [9].Recently the ETosis mechanism was characterized in invertebrates [8,10].The aim of this study was to characterize for the first time gastropod's Haemocytes Extracellular Traps (HETs) induction and formation against metastrongylid larvae.Haemocytes from the slugs species Arion lusitanicus and Limax maximus, and the Giant African Snail Achatina fulica, were cultured with Angiostrongylus vasorum, Crenosoma vulpis, Aelustrongylus abstrusus and Troglostrongylus brevior L1 larvae as well as L3 larvae of A. vasorum at room temperature (±20 °C) and the incubation time varied between 30 min.to 24 h.The visualisation of the HETs was performed using phase contrast microscopy, scanning electron microscopy and fluorescence microscopy.Confronting gastropod haemocytes with the above mentioned species of lungworm larvae revealed in the phase contrast microscopy that L1 and L3 got entangled with a non-defined material originating from the haemocytes.These delicate non-defined ET-like structures were examined in more detail with the technique of SEM imaging.This method renewed our strong suspicion that these structures represent gastropd-derived ETs.Immunofluorescence microscopy revealed that these structures contain histones and DNA.Which have been proven to play an important role in the process of ETosis [11,12].This survey represents first indications on slugs and snails casting HETs.All methods used to visualize Extracellular Trap-formation provided strong evidence that this innate immune defence mechanism also exists in gastropods.

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Assessment of recovery rates and morphology of larvae A. abstrusus in flotation methods using five solutions with different specific gravities (S.G.) Klaudiusz Szczepaniak 1 , Krzysztof Tomczuk The most prevalent cardiopulmonary nematodes in domestic cats in Europe is Aelurostrongylus abstrusus [1].Recently, Baermann migration method is the gold standard for the diagnosis of lungworms invasions, but takes 24 h and requires at least 4 g of fresh fecal samples.Furthermore, taxonomical classification of motile larvae may be difficult, because their identification is particular based on the tail shape [2].Unlike the Baermann method flotation-based techniques are easy to performed, fast and allow to detected wide range of parasites.Fecal samples could be delivered to the laboratory preserved or frozen.Specific gravities of different flotation fluid as well as exposures time resulted in the number and morphologic deformations of the larvae derived from each methods modification [3].The aim of this study was to assessment of the recovery rates and morphology of larvae A. abstratus in flotation methods using five fluids with different specific gravities (S.G.) Fresh fecal sample (6 g) from natural infected with A. abstrusus cat (three years old, male, previously not treated) were obtained.The number of lungworm larvae per gram of feces (LPG) was estimated -2800/g, using modified Baermann methods and McMaster chambers.Subsequently, five flotation with different flotation solutions respectively: 33% ZnSO 4 (SG 1.18) , saturated NaCl (SG 1.20), commercially available NaNO 3 -Fecasol (SG 1.20), saturated NaCl and sacharose (SG 1.25), saturated NaNo 3 (SG 1.33) were performed.We used the following procedure: 1 g fecal sample was mixed with 35 ml flotation solution and poured through a strainer into a (25 ml) glass Erlenmeyer Flask.Slides were directly analyzed under light microscope with Nomarski contrast.A total number of larvae (recovery rates) for each flotation was estimated.Simultaneously larvae were recorded as identifiable (tail was visible) or unidentifiable (tail was not visible e.g., morphologic deformations or curled larvae).Statistical data analysis was performed using Analysis ToolPak Microsoft Office Excel.The larvae of A. abstrusus were found in all flotations.The biggest recovery rates 3.2 and 3.1 were achieved using flotation solutions with the highest specific gravities (saturated solutions of: NaCl/sacharose and NaNo3).In solutions with SG from 1.18 to 1.2 number of detected larvae were lower but their characterized by high percentage of identifiable larvae ranged from 56.3% (saturated NaCl), 71.4% (Fecasol) to 85.7% (33% ZnSO4).Details of results are presented in Table 2.
Aelurostrongylus abstrusus is considered the most prevalent lungworm worldwide in domesticated cats [1,2].High prevalence rates have especially been found in southern Europe [3,4] and studies have indicated that the infection is of clinical relevance [5,6,7].A recent study revealed a high occurrence of A. abstrusus in euthanized cats from eastern Denmark [8] which raised concern of an underestimated national prevalence.Based on these findings, the objective of the present study was to investigate the national prevalence of A. abstrusus in Danish cats.For this purpose, faecal samples from 327 cats were collected between August and October 2015.The study population consisted primarily of outdoor cats from shelters distributed across Denmark and a modified Baermann method was used to test for the infection.The national prevalence of A. abstrusus was 8.3% [95% CI: 5.6-11.9]with substantial regional variation.In Northern Jutland the prevalence was 0% [95% CI: 0.0-8.8]while a prevalence of 31.4% [95% CI: 16.9-49.3]was found in Western Jutland.The prevalence in the remaining regions varied from 4.5-9.7%.Living in rural areas was identified as a risk factor for infection with A. abstrusus (p = 0.0001) and this accounted for most of the variation in regional prevalence.Aelurostrongylosis was not detected in cats younger than 11 weeks and the prevalence in this age group was significantly lower than in older cats (p = 0.002).
Based on these findings, lactogenic transmission seems unlikely, despite the fact that this route has been suggested for the closely related feline lungworm Troglostrongylus brevior [9].The results of the present study demonstrated that A. abstrusus is endemic in Denmark.Therefore, this parasite should be considered an important differential diagnosis in any Danish cat displaying respiratory symptoms.The infection is especially relevant in outdoor cats living in rural areas.Other than rural origin, differences in regional prevalence may result from factors influencing the presence of intermediate and transport hosts, such as climate.However, socioeconomic differences between regions may also in part explain the differences in the current prevalence rates.With increased movement of pets, more extensive testing for A. abstrusus is warranted to monitor the distribution and prevalence of A. abstrusus.
In dogs Angiostrongylus vasorum often causes a severe infection characterized by varying signs similar to those of other canine diseases.
Although this parasitosis may be life-threatening, dog angiostrongylosis is often underestimayed and veterinarians do not use appropriate diagnostic tests.Six cases of angiostrongylosis are here described, with a focus on their clinical features, that were unusual and confounding.Although the six were refereed with clinical signs that may occur in the infection by A. vasorum, the animals were suspected to have other diseases before a correct diagnosis was achieved.Case 1: a dog showed clinical, radiographic and ultrasound features compatible with a pulmonary tumour; case 2: this dog was simultaneously infected by A. vasorum and Dirofilaria immitis but the former nematode was not included in the differential diagnosis ; case 3: a critically ill dog was referred for a severe and then fatal dyspnoea of initially unknown origin; case 4: a thrombocytopenia recorded in a dog with hemorrhages and ecchymoses was erroneously attributed to an inherited, immune-mediated or infective origin; Case 5: a discospondylitis was considered to be the cause of neurological signs in a dog; case 6: a cardio-pulmonary dirofilariosis was diagnosed in a dog that had, on the contrary, only angiostrongylosis.A prompt administration of a parasiticide (in most cases topical moxidectin) was efficacious in the treatment of A. vasorum infection in dogs n. 1, 2, 4 and 5, i.e. those animals that did not show hazardous lung haemorrhages yet at the referral.Currently, canine angiostrongylosis is spreading in various regions for different biological and epizootiological factors.Importantly, animals n. 1-5 were diagnosed with A. vasorum in italian regions where this parasite is not considered endemic.Thus, it is of importance that practitioners must include A. vasorum in the differential diagnosis of any clinical picture compatible with dog angiostrongylosis also when the parasite is not expected to occur and/or in the presence of compatible signs even if the clinical picture is atypical.These clinical cases are described in detail in ref. [1].
). Number of diagnosed cases of canine dirofilariosis per year in Austria of D.
2,3,4, Ana Cristina Oliveira5, Sara Granada5, Ana Patrícia Lopes4,6, Silvana Belo 7 , Luís Madeira de Carvalho 1 , However, data on cardiopulmonary dirofilariosis, a zoonotic mosquito-borne disease that is potentially lethal to companion animals, is non-existent.To assess the potential occurrence of infection with Dirofilaria immitis in canids, 103 domestic dogs presented to a veterinary medical centre in Luanda were evaluated.Luanda was chosen as it is both the capital and the largest city in the country.
Various studies had reported several species of mosquitoes infected by D. immitis larvae such as Haplotype H1 of Culex pipiens in Spain; Cx. pipens complex in Italy, Turkey, Germany and Belarus Republic; Cx. torrentium in Germany and Belarus; Cx. theileri in Madeira (Portugal) and Canary Islands (Spain); Aedes albopictus, Ae. caspius and Coquillettidia richiardii in Italy; Anopheles maculipennis in Italia; 1, Ivan Fenjac 1 , Aleksandar Potkonjak 2 , Suzana Otasevic3, Sara Savic4The first published research on Dirofilaria immitis (D. immitis) infections in Serbia was in the 1990s, when the first cases were determined in dogs, discovered as a side finding during dissections.So far, after many studies, it can be pointed out that Vojvodina, (Northern Serbia) is an endemic region for dirofilariasis in dogs caused by D. immitis.During the period of the last 10 years, prevalence of D. immitis infection in dogs went from 7% to 26,9%.Today, clinical symptoms in dogs can be observed, a regular health check-up in dogs is provided by the veterinary service.Herein we report a very high prevalence of D. immitis infections in dogs from one dog shelter with a total of 19 dogs near Novi Sad, Vojvodina and good outcomes after Ivermectin therapy.The shelter is situated 20 km away from the city of Novi Sad, close to the river Danube, with a lot of trees and grass surfaces around.Out of a total of 19 dogs, 13 dogs had D. immitis infections which were diagnosed at clinical examinations (dogs presented cough and weakness during the regular everyday activities), snap test (SNAP 4DX Idexx) and by Knott test for detection of microfilariae in peripheral blood.In all infected dogs, therapy was started with Ivermectin, with a dose of 0.6 mg/kg per body mass every week for 4 weeks, then every two weeks, followed by once per month.The monitoring of therapy effectiveness was performed every month in all of the dogs due clinical examination and Knott testing.After therapeutic procedure, microfilariae were not detected in blood of all cured dogs.Therapy with Ivermectin and Knott test were repeated for the next 6 months and there were no parasitological positive findings of dirofilariosis.In addition, eight months after the first therapy was given to all of the dogs, parasitological and clinical examinations showed that all dogs were without clinical symptoms and using Knott test microfilariae were not found in blood of examined dogs.
1 Private veterinary practice "Pedigre", Novi Sad, Serbia; 2 Department of Veterinary medicine, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia; 3 Department of Microbiology and Immunology, Medical faculty, University of Nis, Public Health Institute Nis, Nis, Serbia; 4 Scientific Veterinary Institute "Novi Sad, Novi Sad, Serbia Correspondence: Sara Savic (sara@niv.ns.ac.rs)Parasites & Vectors 2016, 10(Suppl 1):A24Fig.5 (abstract A21).See text for description 3 IDEXX Laboratories, Inc., Westbrook, Maine 04092, USA Correspondence: Elias Papadopoulos (eliaspap@vet.auth.gr)Parasites & Vectors 2016, 10(Suppl 1):A25 1, Vesna Lalosevic 1 , Aleksandar Naglic 2 , Stanislav Simin 1 , Ljiljana Kuruca 1 , Aleksandar Spasovic3 1Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, Novi Sad, 21000, Serbia; 2 JKP "Zoohigijena i veterina", Novi Sad, 21000, Serbia; 3 PVA "Mama", Belgrade, 11000, Serbia Correspondence: Ljubica Spasojevic Kosic (ljubicask@polj.uns.ac.rs, ljubica.spasojevic@gmail.com)Parasites & Vectors 2016, 10(Suppl 1):A26 1 , Grzybek Maciek 1 , Andrzej Junkuszew 2 , Paulina Dudko 2 , Pantchev Nikola 3 , Stefaniak Marzena 4 , 4Lubelskie Centrum Małych Zwierząt, ul.Stefczyka 11, 20-151 Lublin, Poland Correspondence: Tomczuk Krzysztof (krzysztof.tomczuk@up.lublin.pl)Parasites & Vectors 2016, 10(Suppl 1):A27 recent works have focussed on various aspects of the infection.First of all, the expansion of A. vasorum in dogs and in wildlife in Europe seems to persist.New reports include cases in dogs from Belgium, Portugal, Bulgaria and Slovakia, all surrounded by countries where A. vasorum had previously been observed.In parallel, studies in wildlife have confirmed that foxes represent the most important reservoir, with prevalences over 70%.Recent epidemiological studies in foxes and dogs showed that annual precipitation and temperature influenced the distribution of A. vasorum, and that in the Alps, altitudes above 700 m asl represent a limiting factor for parasite transmission.Field studies illustrated the variability of spatial distribution and the variability of the slug fauna acting as intermediate hosts, which was suggested to explain the clumpy distribution of A. vasorum.An additional confounder may be represented by birds: in addition to previously described frogs, experimental studies have shown that chicken (and therefore potentially other bird species), may also act as paratenic hosts.In dogs, the classical larval detection in faeces is frequently complemented with PCR performed on different substrates, including bronchoalveolar fluid.Comparisons performed between coproscopic, biomolecular and serological methods testify to the high performance of serological methods.A commercially available test kit for A. vasorum antigen detection allows the diagnosis of canine angiostrongylosis within 15 minutes.It also proved highly sensitive when analysing cardiopulmonary tissue fluid of foxes.Last but not least, the broad variety of clinical signs associated with A. vasorum infection for an excellent camouflaging of the disease, including manifestations in the eyes, neurological disorders, bleeding from various surfaces or internally or even by nematode dermatitis, hepatic abnormalities or concurrent infections with the heartworm Dirofilaria immitis.In clinical patients with respiratory distress the occurrence of pulmonary hypertension was proposed as a negative predictor of survival to the infection Importantly, bleeding seems to occur in up to one third of clinical cases, however results of tests evaluating the coagulation system are not fully consistent and the reasons behind the impaired coagulation are still debated.using the techniques of artificial digestion and microscopy.The confirmation of the lungworm species will be made by specific PCRs.Preliminary data revealed a total A. vasorum prevalence of 4.6% in slugs based on microscopic analyses.The number of A.
most are missing for Germany, we here conducted an epidemiological survey on slugs in selected regions of Hesse and Rhineland-Palatinate. To account for seasonal variations slugs were collected throughout the season in spring, summer, autumn and winter in four different areas (two spots for Hesse and Rhineland-Palatinate, each) that were previously proven to be hyperendemic for A. vasorum fox infections.Thus, a total of 2701 slugs were collected and examined for lungworm larvae 3 Bayer HealthCare -Animal Health, Copenhagen, Denmark; 4 Institute of Parasitology -University of Zurich, Zurich, Switzerland Correspondence: Giulio Grandi (giulio.grandi@sva.se)Parasites & Vectors 2016, 10(Suppl 1):A31 Geographical distribution of metastrongylid nematodes Angiostrongylus vasorum and Crenosoma vulpis in Slovak wildlifepreliminary study Viktória Čabanová, Zuzana Hurníková, Martina Miterpáková Institute of Parasitology, Slovak Academy of Sciences, Košice, 040 01, .9% (22/317) of the samples examined.Duration of clinical signs prior to diagnosis ranged from 14 -210 days.First-stage larvae of Crenosoma vulpis (4.7%; 15/317), Strongyloides stercoralis (0.6%; 2/317), Filaroides hirthi/Oslerus osleri (0.3%; 1/317) and Aelurostrongylus abstrusus (0.3%; 1/317) were detected on Baermann examination.Detection of A. abstrusus L1 in the one dog was considered a spurious finding.Eggs of Paragonimus kellicotti (0.6%; 2/317) and Eucoleus boehmi (0.3%; 1/317) were detected on centrifugal flotation.All of the C. vulpis infections were detected from October to May with nearly half occurring in March.Baermann examination of the 3-day composite sample detected 86.7% (13/15) of the C. vulpis infections compared to 73.3% (11/15) detection by examination of a single (day 3) sample.Larval shedding levels ranged from 0 -455 LPG (Mn = 22.2 LPG); only 2 dogs shed more than 20 LPG.Lungworm infection should be considered as a possible cause in any case of respiratory disease in dogs in eastern Canada (and likely elsewhere).Three daily Baermann fecal examinations had greater C. vulpis detection sensitivity than a 3-day collection composite and both were superior to examination of a single day collection sample.
vasorum and C. vulpis.Rates of infection with A. vasorum and C. vulpis and their geographical distribution were analysed and proved statistically.From 2003 to 2015 A. vasorum and C. vulpis were detected in 477 (0.9%) and 248 (0.5%) of 54,934 examined dog samples, respectively.The percentage of A. vasorum positive dogs increased2004 - 2006, 2007 -2009, 2010 -2012, and 2013 -2015 from 0.1%, 0.7%, 0.9% to 1.8%, respectively.In 2014 and 2015 the rates of infection with A. vasorum were significantly higher (p < 0.05) compared to each year in 2003 -2007 and in 2003 -2010, respectively.There were no statistically significant differences between the rates of infection with C. vulpis of every year in 2003 -2015.Most of the infected dogs with A. vasorum and C. vulpis were found in south-western Germany.Clusters of infections with A. vasorum were located in Baden-Wuerttemberg, Saarland, North Rhine-Westphalia and Rhineland-Palatinate. Our data and the expanding range from which canine cases are reported are consistent with the hypothesis that A. vasorum is spreading.Crenosoma vulpis were detected in 1 control and 8 symptomatic dogs respectively.In the latter group one dog was found seropositive for A. vasorum but only C. vulpis larvae were found via the Baermann technique.All seropositive and symptomatic dogs (n = 19) exhibited cardio-pulmonary symptoms.In conclusion this seroepidemiological study demonstrated a fairly high seroprevalence in Southern Belgium for A. vasorum.The Angio detect™ IDEXX was found to be highly suitable in this context as the sampling, preservation and examination of stools were difficult and somewhat unreliable in the field.However, coproscopy remains a useful tool in dogs infected for less than 9 weeks and for the identification of other canine lung nematodes such as Crenosoma vulpis.
Oslerus rostratus, Troglostrongylus spp.) by the ingestion of infective third-stage larvae (L3) in the tissues of gastropod intermediate hosts (IMH) and in some species also paratenic hosts.Speculation on potential exposure due to L3 shed into the environment has arisen due to reports of L3 released by laboratory aquatic snails experimentally infected with A. abstrusus, A. vasorum and Troglostrongylus brevior.Spontaneous shedding has not been reported for these species in the terrestrial gastropod natural IMH (slugs, land snails).First-stage larvae of A. abstrusus, A. vasorum and C. vulpis were each placed on lettuce (400 -1445 L1/slug) and fed to laboratory raised Limax maximus in separate exposure groups of 12-42 slugs.Anastasia Diakou 1 , Despina Migli 2 , Angela Di Cesare 3 , Dimitra Psalla 4 , Dionisios Youlatos 2 , Donato Traversa 3 Thessaloniki, 54124, Greece; 2 Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece; 3 Faculty of Veterinary Medicine, Teaching Veterinary Hospital, Località Piano d'Accio snc, Teramo, 64100, Italy; 4 Laboratory of Pathology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece A39Angiostrongylus chabaudi: first description of the diagnostic stage and confirmation of European wildcat as definitive host 1 Laboratory of Parasitology and Parasitic Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Correspondence: Anastasia Diakou (diakou@vet.auth.gr)Parasites & Vectors 2016, 10(Suppl 1):A39

Table 2 A
Comparison of various flotation fluids in A. abstrusus larvae (L1) detection First report of Angiostrongylus vasorum infections in dogs as well as in the neozoan intermediate host (Achatina fulica) in Medellín, Colombia Felipe Penagos 1,2 , Jesed Gutiérrez 1 , Juan D. Velez 1 , Diego Piedrahita 1 , Malin Lange 2 , Carlos Hermosilla 2 , Anja Taubert 2 , Jenny Chaparro 1 1 CIBAV research group, Veterinary Medicine School, Faculty of Agrarian Sciences, University of Antioquia, Medellín, Antioquia, 050034, Colombia; Institute of Parasitology, Justus Liebig University Giessen, Giessen, 35392, Germany Correspondence: Felipe Penagos (Felipe.penagos@udea.edu.co)Parasites& Vectors 2016, 10(Suppl 1):A46Angiostrongylus vasorum is considered as one of the most pathogenic species of the cardiopulmonary system of wild and domestic canids worldwide.As such, in 1961 this metastrongyloid parasite was detected in South America in crab-eating foxes (Cerdocyon thous)i n Colombia and in domestic dogs in Brazil.These reports in demonstrated clearly the presence of this parasite in South America.Nonetheless, since then very little has been published on A. vasorum infections neither in wild canids nor domestic dogs in South America and Colombia.Thus, aim of this study was to gain current knowledge on the presence of A. vasorum in domestic dogs as well as neozoan intermediate hosts by analysing dog faecal samples collected in public parks in Medellin city and from collected neozoan terrestrial snails (Achatina fulica).In total 364 faecal samples were collected from February to April 2016, analysed thereafter by Bearmann funnel test with submersion of the samples for at least 24 h.Additionally 300 A. fulica snails were collected and digested for the presence of A. vasorum larvae The larvae obtained from molluscs were identified on the basis of 2