Autochtonous infection of dogs and slugs with Angiostrongylus vasorum in Hungary

On the course of a helminthological survey of the dogs of Baranya County, Hungary Angiostrongylus vasorum infection was detected in two asymptomatic dogs. Identification of the parasite was based on morphology of the first-stage larvae (L1) isolated from droppings, and successful experimental infection with first stage larvae to laboratory reared Discus rotundatus and Lissachatina fulica snails, in order to exclude species of the family Filaroididae that have similar larvae to A. vasorum. While angiostrongylosis is widespread among foxes, this is the first report of A. vasorum infection in housedog in Hungary. In gardens, where infected dogs were being kept 91 specimens of 6 species of limacid and arionid slugs were collected of which 5 specimens of Arion lusitanicus were found to carry larvae of A. vasorum. Dogs usually do not ingest such large slugs willingly. Frogs are known to act as paratenic hosts in the life cycle of A. vasorum. Since one of the infected dogs harboured also infection with the intestinal trematode Alaria alata, of which frogs certainly play the role of the second intermediate host, therefore it is assumed that in this case the dog became infected with A. vasorum by eating frogs.     

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.     

Serologic detection of Angiostrongylus vasorum infection in dogs

Angiostrongylus vasorum, French Heartworm, is a metastrongylid nematode infecting the pulmonary arteries and right heart of wild and domestic canids in various regions of the world. Infection in dogs can result in fatal cardiopulmonary disease. A single endemic focus of A. vasorum in North America occurs in the southeastern portion of Newfoundland, Canada. Dogs are currently diagnosed by detection of first-stage larvae shed in feces using the Baermann technique or fecal flotation. However, these procedures may lack sensitivity due to intermittent fecal larval shedding. The potential for using detection of circulating worm antigen for diagnosis was investigated by developing a sandwich-ELISA using rabbit anti-whole adult worm antiserum. This test detected circulating antigen in sera from 22/24 Baermann positive dogs naturally infected with A. vasorum. Negative results (0/52) were obtained from sera collected from Baermann negative dogs from outside of the endemic region, and from sera (0/30) from dogs from non-endemic regions that were infected with Crenosoma vulpis, the fox lung worm. Receiver operating curve analysis gave a specificity of 100% and a sensitivity of 92% for the sandwich-ELISA at an optical density cut-off of 0.19. Subsequently, 239 dogs from Newfoundland displaying clinical signs of cardiopulmonary disease, were examined using both the Baermann fecal examination and the sandwich-ELISA. Larvae were detected in 10% (24/239) of these dogs by fecal examination, whereas the sandwich-ELISA detected circulating antigen of A. vasorum in serum from 18.8% (45/239) of the dogs. This suggests that fecal diagnostics may have missed approximately half of the A. vasorum infected dogs, and that the sandwich-ELISA may be a useful tool in the diagnosis of this parasite.     

Cytological Analysis of Bronchoalveolar Lavage Fluid in the Diagnosis of Spontaneous Respiratory Tract Disease in Dogs: A Retrospective Study

Results of cytological analysis of bronchoalveolar lavage (BAD fluid were compared with clinical diagnoses in dogs that presented with signs of respiratory disease to referral hospitals. Of 68 dogs in which a clinical diagnosis was possible, BAL cytological findings were considered definitive for the diagnosis in 17 cases (25%), supportive of the diagnosis in 34 cases (50%), and not helpful in 17 cases (25%). Findings were most often considered supportive of or definitive for the clinical diagnosis in dogs with alveolar or bronchial radiographic patterns, or the presence of pulmonary masses. BAL results among lung lobes differed in 23 of 63 dogs (37%) with diffuse radiographic patterns. Tracheal wash cytology differed from BAL fluid cytology in 45 of 66 dogs (68%). Bronchoalveolar lavage was a clinically useful procedure for the diagnostic evaluation of dogs with signs of respiratory disease.     

An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs

Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretory - antigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1-99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5-98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n=23), Crenosoma vulpis (n=14), Ancylostoma caninum (n=4) or Toxocara canis (n=20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88-92 dpi, OD values decreased within 13-34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations.     

Clinical findings,bronchoalveolar lavage fluid cytology and matrix metalloproteinase-2 and -9 in canine pulmonary eosinophilia

We characterized clinical and clinicopathological features, and the involvement of gelatinolytic matrix metalloproteinases (MMP-2 and -9) in canine pulmonary eosinophilia (PE). Study material consisted of 20 PE dogs and 16 healthy beagles. All dogs underwent a similar clinical examination and bronchoalveolar lavage (BAL). Analysis for cell count and differential cell count of BAL fluid (BALF), arterial blood gas analysis before and after BAL, and thoracic radiographs before BAL and after treatment were obtained. Twelve dogs were re-evaluated and six relavaged. MMP-2 and MMP-9 in BALF were analysed by zymography, Western immunoblotting and immunocytochemistry.In the PE dogs, BALF, cell count, number and percentage of eosinophils, and numbers of macrophages, lymphocytes, neutrophils, mast cells and epithelial cells were all significantly elevated. Blood eosinophilia was detected in half of the PE dogs. Three PE dogs had mild hypoxaemia. The BAL procedure had an equal effect on PE and healthy dogs' arterial blood gas values. Bronchointerstitial densities were seen in PE dogs' radiographs. Treatment of PE decreased BALF cell count, eosinophil count and percentage and diminished radiographic changes. Gelatinolytic activity was higher in PE dogs' BALF. BALF macrophages and epithelial cells were the principal sources of the MMP-9.     

Collection and interpretation of respiratory cytology

Advances in pulmonary cytological sampling have helped establish a more accurate diagnosis and better therapeutic choices for the respiratory patient. Choosing the appropriate test is necessary to maximize the potential diagnostic yield and should be based on the clinical presentation of the patient as well as the minimum database. This article describes several methods of collection of cytological samples for the canine and feline upper and lower respiratory tracts, defines normal respiratory cytology, and discusses normal cytological characterization of various respiratory diseases. Categorization of cytological findings may aid the clinician in narrowing the potential etiologies for the pulmonary disorder. The authors focus on cytological recovery and analysis from transtracheal wash (TTW), bronchial brushing, transthoracic lung aspiration (TTLA), bronchoalveolar lavage (BAL), and open chest biopsy. Indications, complications, and contraindications for each procedure are discussed. Variation in expected findings among BAL, TTW, TTLA, and bronchial brushings are described. Appropriate sampling technique, transport, and processing are also emphasized.     

Coagulation abnormalities associated with acute Angiostrongylus vasorum infection in dogs

Ten dogs were experimentally infected with 150 3rd-stage larvae of the lungworm Angiostrongylus vasorum (Baillet, 1866). Blood samples obtained once a week for 5 weeks after infection was induced were examined for coagulation alterations. Thrombocytopenia developed in infected dogs before and after parasitic patency, which occurred 6 weeks after infection was induced. Prolongation of activated partial thromboplastin time and thrombocytopenia initially were associated with periods of larval migration and, later, at patency, with pulmonary egg embolism and hatching of 1st-stage larvae in the lung. Decreased factor-V activity, significantly shortened values for the prothrombin time, and increased factor-VIII activity were evident during postpatent periods of parasitic infection. Significant changes were not found in factors-XII and -IX activities throughout the course of infection. Coagulation abnormalities were attributed to excessive intravascular coagulation. Immature adults and first stages of the parasite seemed to have initiated the coagulopathy in the prepatent phase of infection, with subsequent coagulation abnormalities occurring at the time of patency.     

Pathological findings in dogs naturally infected with Angiostrongylus vasorum in Newfoundland and Labrador, Canada.

Fifty-six dogs from St. John's, Newfoundland, Canada, were evaluated for Angiostrongylus vasorum infection. Small numbers of nematodes were found within pulmonary arteries of 6 dogs. Larvae were identified in fecal samples in 2 of 6 dogs. All 6 dogs had multifocal granulomatous pneumonia and sometimes foci of chronic thrombosis, which varied from very mild to severe. One dog had extensive pulmonary lesions resulting in cor pulmonale. Right heart failure was characterized by right ventricular hypertrophy, hepatic congestion, ascites, and hydrothorax. Microscopically, in most cases, eggs, larvae, and sometimes intravascular adults, were present within lung tissue sections. Small foci of granulomatous inflammation with and without larvae were present in kidney and brain in 4 dogs. An additional dog, diagnosed antemortem with angiostrongylosis via fecal examination, was also examined. Pathological findings consisted of severe pyogranulomatous interstitial pneumonia with myriad eggs, larvae, and numerous intravascular pulmonary adult nematodes with extensive arterial thrombosis. Five hundred and seventy-two adult worms were removed from pulmonary arteries. Foci of granulomatous inflammation, often associated with larvae and/or eggs, were present in tracheobronchial lymph nodes, adrenal gland, brain, and kidneys. Severe seizuring noted antemortem was attributed to several large, discrete areas of acute hemorrhagic infarction within the cerebrum and cerebellum. Natural A. vasorum infection in domestic dogs in eastern Newfoundland causes lung pathology of variable severity, which in some cases, may progress to cor pulmonale and which may be associated with extrapulmonary lesions and clinical signs.     

Larval output of infected and re-infected dogs with Angiostrongylus vasorum (Baillet, 1866) Kamensky, 1905

Canine angiostrongylosis is a nematode infection in domestic dogs and wild canids. A natural infection in a domestic dog frequently leads to pneumonia, loss of physical performance, coughing, anemia, cardiac insufficiency, pulmonary fibrosis and death. The main diagnostic method is based on the finding of Angiostrongylus vasorum first-stage larvae (L1) in infected dog feces. With this objective, 11 experimentally exposed to 100 third-stage larvae (L3) per kilogram of body weight (mean = 885.45 L3/animal; S.E. = 77.7). The animals were monitored for 300 days post-single-infection (PI) and the quantity of L1 output measured. Our results showed an irregular excretion of L1 and a variation in the pre-patent period (33-76 days) and the number of L1 excreted by individual animals (1-1261 L1/g). After 300 days PI, five dogs were exposed a second time and monitored for 300 days post-re-infection (PRI) (=600 days PI). The quantity of L1 output demonstrated that double exposed dogs also presented an irregular excretion of L1 but a smaller variation in the number of L1 excreted by individual animals (4-550 L1/g).     

Hematological and coagulation profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866).

Hematological and coagulation profiles were studied in crossbred dogs experimentally infected with Angiostrongylus vasorum. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L(3)) of A. vasorum per kilogram of body weight. A third group of five uninfected animals was used as control. One sample of 10 ml of blood was collected from each animal on the 10, 20, 30, and 45 days after inoculation (dai) and at 30-day intervals thereafter for the remainder of the 210-day experimental period. The blood sample was used for the complete hemogram and platelet count, as well as measurements of prothrombin time, partial thromboplastin time and factors V and VIII. Anemia was observed in infected dogs, 6 weeks after the infection. The eosinophils presented peaks in four periods after infection. Thrombocytopenia became accentuated on the 72 dai. Decreased prothrombin time activity and increased partial thromboplastin time were observed at the 6 and 9 weeks after infection and decreased of factors VIII and V activities occurred from 4 to 6 weeks after infection. It may be conclude that infection by A. vasorum in dogs may cause a discrete anemia during the acute phase which is probably regenerative. In addition, important hemostatic alterations due to the infection suggest a chronic intravascular consumption coagulopathy.     

Biochemical serum profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866)

The biochemical profiles of crossbred dogs experimentally infected with the parasite Angiostrongylus vasorum were studied. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L3) of A. vasorum per kilogram of body weight. A third group of five uninfected animals were used as control. Serum from these animals were used for biochemical tests to measure total and fractioned proteins, urea, creatinine and to determine the activities of aspartate (AST), alanine (ALT) aminotransferase, gamma-glutamyl transferase (GGT), alkaline phosphatase (PAL) and creatine kinase isoenzyme MB (CK-MB). The alpha-1, alpha-2 and beta-globulins fractions showed alterations during acute phase of the infection. No modifications were observed in the biochemical profiles of ALT, AST, GGT, PAL, urea and creatinine. CK-MB was shown to be a good early indicator of cardiac injury in dogs experimentally infected with A. vasorum.     

Brain and spinal cord haemorrhages associated with Angiostrongylus vasorum infection in four dogs

Multifocal haemorrhages associated with Angiostrongylus vasorum infection were observed in the central nervous system of four dogs with neurological signs including depression, seizures, spinal pain and paresis. In magnetic resonance images the majority of the lesions were isointense or slightly hyperintense in T1-weighted images, hyperintense in T2-weighted images and hypointense in T2*-weighted (gradient echo) images, compatible with haemorrhages more than seven days old. Lesions were found in the brain of three of the dogs and in the spinal cord of two. The cerebrospinal fluid contained high concentrations of protein and evidence of erythrophagia. All the dogs had coagulopathy and pulmonary haemorrhage of varying severity. A vasorum larvae were detected in the faeces of each of the dogs. Neural A vasorum was confirmed at postmortem examination in two dogs.     

Canine angiostrongylosis: the French heartworm: an emerging threat in North America

Angiostrongylus vasorum, French heartworm, is a metastrongloid parasite found in the pulmonary arteries and right ventricle of wild and domestic canids and various other animals. The natural definitive hosts are species of foxes. The geographic distribution of the parasite includes various countries of Europe, Africa, South America, and North America. Angiostrongylosis is considered an emerging disease in dogs in Europe. In North America, autochthonous A. vasorum infection occurs only in the Canadian province of Newfoundland-Labrador. Computer modeling suggests there is a high probability that A. vasorum will spread to other parts of North America and will likely become endemic in the eastern half of the continent and in the states and provinces along the western coast. Animals acquire infection by the ingestion of gastropod or frog intermediate hosts that carry the infective 3rd-stage larvae. Frogs can also serve as paratenic hosts. Definitive antemortem diagnosis is by detection of L(1) in feces, sputum, or bronchoalveolar lavage samples. Baermann fecal examination is the most reliable method for fecal detection. However, false negative results can occur due to the typical erratic/sporadic fecal larval shedding pattern of A. vasorum. Recently, promising new methods for A. vasorum infection diagnosis have been reported involving polymerase chain reaction of blood and fecal samples and a sandwich ELISA for detection of circulating worm excretory/secretory antigen. Current treatment options include moxidectin, milbemycin oxime, and fenbendazole.     

Haemothorax associated with Angiostrongylus vasorum infection in a dog

Angiostrongylosis was diagnosed in a dog presenting with haemothorax on the basis of detection of Angiostrongylus vasorum first-stage larvae both in the pleural effusion and in faeces. A one-year-old, male, mixed-breed dog was presented with fever, depression and persistent cough of one month's duration. Clinical examination revealed temperature of 39.5 degrees C, loud bronchovesicular sounds on thoracic auscultation and attenuated cardiac sounds. Thoracic radiographs showed a moderate bilateral pleural effusion and a diffuse interstitial pulmonary pattern, with an alveolar pattern in one lobe. Routine haematology revealed anaemia and leucocytosis with eosinophilia, basophilia and thrombocytopenia. Coagulation assays showed a consumptive coagulopathy resembling disseminated intravascular coagulation. The relationship between haemothorax and the presence of A vasorum larvae in the pleural effusion is discussed. The dog was successfully treated with fenbendazole until negative for larvae on faecal examination. This case report indicates that A vasorum infection should be considered as a possible aetiological cause of haemothorax in dogs.     

Angiostrongylus vasorum causing meningitis and detection of parasite larvae in the cerebrospinal fluid of a pug dog

This case report describes the presence of Angiostrongylus vasorum larvae in cerebrospinal fluid in an 11-month-old pug dog and the relative magnetic resonance images compatible with a focal meningitis. Clinical signs were compatible with a cerebellar lesion, and diagnosis was confirmed by parasitological analysis on faecal and endotracheal lavage samples. Treatment with fenbendazole and prednisolone resulted in a complete resolution of the clinical signs in two months time. A vasorum infection should be considered a possible aetiology of intracranial inflammation in dogs.     

Haematological and biochemical changes in dogs naturally infected with Angiostrongylus vasorum before and after treatment

Haematological and biochemical parameters were studied prospectively in 48 dogs naturally infected with Angiostrongylus vasorum in a primary care setting. Samples for analysis were obtained when treatment was started and 42days afterwards. Prior to treatment, 21% of affected dogs exhibited eosinophilia, whereas increased total white blood cell (WBC) counts and neutrophilia were observed in only 4.2%. WBC counts and concentrations of neutrophils, eosinophils and monocytes decreased significantly from days 0 to 42, indicating that, even in dogs without elevated absolute blood values, a low grade inflammatory response may be present in dogs with A. vasorum infection. Biochemical changes (especially an increase in serum globulins and a decrease in serum fructosamine) were in agreement with the findings of other studies. The results show that the diagnosis of canine angiostrongylosis should not be excluded based on unremarkable haematological and blood biochemical parameters. They also support our recent finding that a low serum fructosamine concentration may be associated with infection with A. vasorum.     

Serum fructosamine concentrations in 59 dogs naturally Infected with Angiostrongylus vasorum

Retrospectively, 89 cases of dogs infected with Angiostrongylus vasorum were examined. Fifty-nine of these 89 dogs fulfilled the criteria of not being dually infected with Crenosoma vulpis as well as having a full biochemistry profile including serum fructosamine available. The mean serum fructosamine value of the 59 dogs was 236 micromol/l (reference value 258-348 micromol/l) and significantly lower than the serum fructosamine level of 314 micromol/l in a control group of 42 clinically healthy dogs. Eleven dogs were available for follow up after successful treatment of angiostrongylosis. In this group, the serum fructosamine value rose from a mean of 244 micromol/l to a mean of 320 micromol/l following treatment. Serum glucose, albumin and protein were all within the respective reference ranges at all sampling points. The results indicate that serum fructosamine could be affected by infection with A. vasorum. Furthermore, this change cannot be explained by measurable changes in the level of glucose, albumin or protein. The clinical impact of this study is that a low fructosamine value may indicate infection with A. vasorum thereby suggesting a Baermann test to be performed.     

Prevalence study of the lungworm Aelurostrongylus abstrusus in stray cats of Portugal

In this study we have investigated the prevalence of aelurostrongylosis, one of the most common feline pulmonary parasitic diseases, in cats from the north-west region of Portugal. For this purpose, 97 faecal samples were collected from cats at risk of being infected by Aelurostrongylus abstrusus in an animal shelter and in a municipal facility. Using the Baermann-Wetzel coprological technique, faecal shedding of first stage larvae (L1) was detected in 17.4% of the cats. Based on this result, it can be concluded that this lungworm infection seems to be common among feral cats in the north-west region of Portugal, in spite of the fact that clinical aelurostrongylosis is not frequently diagnosed by feline practitioners in the area. This parasitic disease should be included in the differential diagnosis of cats presenting with coughing or dyspnoea, and it also should be extended to asymptomatic animals with pulmonary nodules detected by image diagnosis.     

PCR检测感染早期小鼠血液中旋毛虫幼虫的研究

为了研究PCR检测感染小鼠血液中旋毛虫DNA的敏感性,应用旋毛虫1.6 kb重复序列为扩增靶序列对旋毛虫(T1)、乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)和南方旋毛虫(T7)肌幼虫DNA进行PCR扩增,并检测小鼠感染20、100、300条T1肌幼虫后不同时间的外周血.结果表明,T1、T4和T7肌幼虫可扩增出特异性目的条带(510 bp),而T2和T3无扩增产物;1、0.04和0.02条T1、T4和T7肌幼虫均能扩增到清晰的目的条带(510 bp).20条幼虫感染小鼠后5 d～6 d,PCR阳性率均为7.69%;100条幼虫感染小鼠后5 d～12 d可检出旋毛虫DNA,其中感染后5 d～7 d的阳性率分别为30.77%、38.46%及30.77%;300条幼虫感染小鼠后5 d～15 d可检出旋毛虫DNA,感染后7 d的阳性率为61.54%,感染后6 d与8 d～10 d的阳性率均为53.85%. 3组旋毛虫感染小鼠PCR阳性率间的差异有统计学意义(p<0.01),PCR阳性率随感染剂量的增加而升高(p<0.01),100条与300条感染小鼠感染后不同时间的PCR阳性率与检测时间有相关性(p<0.01).以上实验结果表明PCR检测感染小鼠血液中旋毛虫DNA的敏感性与感染程度和检测时间有关,对感染早期旋毛虫抗体阴性宿主有一定诊断价值.