The Chronology of Angiostrongylus vasorum (Baillet, 1866), Kamensky, 1905: Infection in Biomphalaria glabrata (Say, 1818).

The intermediate hosts of the French heartworm Angiostrongylus vasorum are aquatic and terrestrial gastropods. The present work is aimed at clarifying the sites of penetration and the migratory routes of A. vasorum in Biomphalaria glabrata snail tissues and evaluating their perilarval reaction with regard to the cellular composition and histological alterations involved in the gastropod response to infection. Biomphalaria glabrata snails were individually infected with 1000 first-stage larvae (L1) of A. vasorum each and killed at predetermined times after infection. Percutaneous infection occurred simultaneously with oral infection. Despite larval tropism to the fibromuscular tissue, some larvae were located in different tissues and organs. A perilarval reaction was observed around the larvae in a fibromuscular layer, appearing later around the larvae located in the viscera. The number of hemocytes surrounding the larvae increased gradually, forming a pregranuloma. Larval death and degeneration were not observed. No defined migratory pattern occurred, and larval development was apparently not associated with particular tissues or organs. In addition, the infection by A. vasorum induces a systemic mobilization of hemocytes in perilarval reaction.

In vitro and in vivo action of Piptadenia viridiflora (Kunth) Benth against Haemonchus contortus in sheep.

Anthelminthic resistant populations of Haemonchus contortus are a major problem in sheep rearing, but plant extracts may offer viable alternative treatments. In our preliminary studies, Piptadenia viridiflora was frequently selected by sheep grazing in the Cerrado. The present research evaluated its in vitro and in vivo anthelmintic activity. The HPLC chromatograms of P. viridiflora aqueous extract (AE) and ethanolic extract (EE) showed the presence of flavonoids. The total condensed tannin (proanthocyanidin) was 0.2 and 1.01% in AE and EE, respectively. In an egg hatching inhibition (EHI) test, the LC90 of AE was 2.4mg/mL, and, of EE, was 2.1mg/mL. After tannin extraction, higher EHI and lower LC90 were observed. In a larval development inhibition test, the LC90 of AE was 13.66mg/g of fecal culture. The highest dose of AE administered to mice (203.0mg/kg bw) was well tolerated, suggesting low toxicity. In vivo, AE was orally administered to lambs at 283mg/kg bw, and, at weeks one, two, and three post-treatment, the mean fecal egg count (FEC) was significantly lower than in untreated lambs (P<0.05). Blood parameters were normal and similar in untreated and treated sheep. For all lamb groups, the mean total serum protein was significantly higher at week two post-treatment than at other evaluated periods (P<0.05). Piptadenia viridiflora extracts had low condensed tannin content and exhibited high anthelminthic efficacy in vitro and significantly reduced FEC. Tannins were not shown to be the principal components affecting EHI, hence it is necessary to isolate and characterize the principal active P. viridiflora compounds, and to assess their possible synergism.

The development of Angiostrongylus vasorum (Baillet, 1866) in the freshwater snail Pomacea canaliculata (Lamarck, 1822).

Angiostrongylus vasorum is a parasitic nematode that infects the heart and pulmonary artery and its branches of domestic and wild canids. The parasite can use several species of terrestrial and aquatic molluscs as intermediate hosts, although susceptibility varies. Pomacea canaliculata is a mollusc found in lakes, swamps and rivers in South America. In this study, we evaluated the susceptibility, parasite growth, oviposition and larval development of 282 P. canaliculata infected with 500 A. vasorum first-instar larvae (L1). From day 5 post-infection (pi) to day 30 pi, seven specimens per day were sacrificed to recover the larval instars. We compared 50 egg masses from infected and uninfected molluscs to determine the number of eggs per clutch, the hatching rate and the growth of the molluscs. The percentage of recovered larvae ranged from 39.17% to 67.5%. First-stage larvae (L1) were found until day 19 pi, second-stage larvae (L2) were found from days 11 to 25 pi, and third-stage larvae (L3) were recovered only after day 19 pi. Infected snails exhibited the most eggs during spawning, although the rate of hatching and shell size were lower in the infected snails compared with controls. This is the first report of an experimental infection of P. canaliculata with A. vasorum, and the results confirm the non-specificity of the nematode in relation to the intermediate host and indicate the importance of epidemiological surveys of this parasite and mollusc.

Gallus gallus domesticus: paratenic host of Angiostrongylus vasorum.

Angiostrongylus vasorum, a parasite of the cardiorespiratory system in canids, has a heteroxenous biological cycle in which the intermediate hosts are terrestrial and aquatic mollusks. Generally, canids become infected by ingesting the intermediate host or paratenic hosts, such as amphibians, that contain infective larvae (L3). However, there are no reports of birds as paratenic hosts of A. vasorum. To evaluate the susceptibility and viability of Gallus gallus domesticus as a paratenic host of A. vasorum, 17 Cobb chickens were randomly divided into two groups. The animals in group A were inoculated with third stage larvae of A. vasorum, and those in group B ate snails inoculated with A. vasorum L3. At 30 days post-infection, the chickens were killed, and the muscles and organs were placed in a pepsin-HCl solution (1% HCl (37%), 1% pepsin) for 3h in an oven at 40°C to recover the L3. In group A, 1863 L3 were recovered per chicken. In group B, 2585 L3 were recovered. A dog that ingested organs and tissues from a chicken from group A released first-stage larvae of A. vasorum in its feces 51 days after infection; the dynamics of this process were monitored for 107 days, when treatment with 25 mg fenbendazole/kg body weight was performed for 21 days. Chickens nourished with infected snails or with infective L3 may be a source of infection for dogs indicate that G. gallus is a potential paratenic host for this parasite.

Rupture of the thoracic aorta associated with experimental Angiostrongylus vasorum infection in a dog.

This note describes the sudden death of a dog by the rupture of the thoracic aorta caused by the presence of Angiostrongylus vasorum. A female mongrel canine with a history of weight loss and exhaustion died two hours after clinical examination. At necropsy, performed one hour after death, showed the presence of clotted blood in the thoracic cavity. Haemothorax was diagnosed. The thoracic aorta wall was thin, congested and an abnormal hole in the wall was detected approximately 0.5 cm from the entrance to the diaphragm. From clotted blood collected from the thoracic cavity, 224 first stage larvae (L1) and 15 adults of Angiostrongylus vasorum were recovered alive. Also, from a blood clot found in the aorta, four adult females and 47 L1 larvae were recovered alive. Possibly, this parasite was responsible for the aortic rupture and death of the animal.

Angiostrongylus vasorum: Experimental Infection and Larval Development in Omalonyx matheroni.

The susceptibility and suitability of Omalonyx matheroni as an intermediate host of Angiostrongylus vasorum and the characteristics of larval recovery and development were investigated. Mollusks were infected, and from the 3rd to the 25th day after infection, larvae were recovered from groups of 50 individuals. The first observation of L2 was on the 5th day, and the first observation of L3 was on the 10th day. From the 22nd day on, all larvae were at the L3 stadium. Larval recovery varied from 78.2% to 95.2%. We found larval development to be faster in O. matheroni than in Biomphalaria glabrata. Our findings indicate that this mollusk is highly susceptible to A. vasorum. Infective L3 were orally inoculated into a dog, and the prepatent period was 39 days. This is the first study to focus on O. matheroni as an intermediate host of A. vasorum.

Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first-stage larvae.

Angiostrongylus vasorum is a nematode that parasitizes domestic dogs and wild canids. We compared the predatory capacity of isolates from the predatory fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34), Monacrosporium sinense (SF53) and Arthrobotrys robusta (I31) on first-stage larvae (L1) of A. vasorum under laboratory conditions. L1 A. vasorum were plated on 2% water-agar (WA) Petri dishes marked into 4 mm diameter fields with the four grown isolates and a control without fungus. Plates of treated groups contained each 1000 L1 A. vasorum and 1000 conidia of the fungal isolates AC001, NF34, SF53 and I31 on 2% WA. Plates of the control group (without fungus) contained only 1000 L1 A. vasorum on 2% WA. Ten random fields (4 mm diameter) were examined per plate of treated and control groups, every 24 h for 7 days. Nematophagous fungi were not observed in the control group during the experiment. There was no variation in the predatory capacity among the tested fungal isolates (P>0.05) during the 7 days of the experiment. There was a significant reduction (P < 0.05) of 80.3%, 74.5%, 74.2% and 71.8% in the means of A. vasorum L1 recovered from treatments with isolates AC001, NF34, SF53 and I31, respectively, compared to the control without fungi. In this study, the four isolates of predatory fungi were efficient in the in vitro capture and destruction of A. vasorum L1, confirming previous work on the efficiency of nematophagous fungi in the control of nematode parasites of dogs and as a possible alternative method of biological control.

Sympatric and allopatric combinations of Lymnaea columella and Fasciola hepatica from southern and south-eastern Brazil.

Experimental infections of Lymnaea columella with Fasciola hepatica were carried out to test the effect of sympatric and allopatric combinations between parasite and host, by using snails and flukes from southern and south-eastern Brazil. Four groups of 50 snails were infected with four miracidia per snail: two groups in sympatric and two groups in allopatric combinations. Sympatric combinations between parasite and host were more efficient than allopatric ones when snails from Itajubá were used, but the opposite was observed in infections involving snails from Pelotas. The sympatric association between L. columella and F. hepatica from Itajubá was significantly higher than in the other combinations. We concluded that the host-parasite relationship between L. columnella and F. hepatica may vary according to the geographical origin of the snails and flukes involved.

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs.

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.

Concordância entre duas técnicas coproparasitológicas para diagnóstico de Fasciola hepatica em bovinos / Evaluation of two available methods to detect eggs of Fasciola hepatica in cattle faeces

Two different methods to detect Fasciola hepatica in faeces using kappa index were compared with four tamises technics and sequencial fitration in two tamises using Flukefinder® apparatus. High accordance between the two methods was observed. The values showed high sensibility and specificity.

Influence of shell size of Lymnaea columella on infectivity and development of Fasciola hepatica.

Experimental infections of Lymnaea columella with Fasciola hepatica were carried out to determine the influence of shell size on the infection rate and on the outcome of rediae and cercariae. Snails were divided into seven groups according to shell size: 2-4 mm, 5-6 mm, 7-8 mm, 9-10 mm, 11-12 mm, 13-14 mm and 15 mm or more. One hundred snails in each group were infected by using four miracidia for each snail. Snails with larger shell size showed a lower infection rate, the groups presenting the highest (79%) and lowest (2%) proportions of positives being those of 5-6 mm and 15 mm or more, respectively. Cercariae were present in 21% of them at 31 days post-infection, and cercarial shedding was observed 61 days post-infection. It was concluded that there is a non-linear negative association between shell size and infection rate.

Development of Fasciola hepatica in Lymnaea columella infected with miracidia derived from cattle and marmoset infections.

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.

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).

Effect of Angiostrongylus vasorum infection on Biomphalaria tenagophila susceptibility to Schistosoma mansoni.

Concomitant infection with different parasites may be a helpful laboratorial strategy leading to the better understanding of the mechanisms used by the internal defense system (IDS) of Gastropoda to deal with helminth infection, such as Schistosoma mansoni. This work reports the effect of co-infection of Angiostrongylus vasorum and S. mansoni in hemocyte activity and in the outcome of infection. The simultaneous infection resulted in an increase of snail susceptibility to S. mansoni. In contrast, snails infected with both parasites, 15 days apart, did not show differences in the susceptibility compared to a single parasite infection. The increased susceptibility was measured by the significantly higher number of migrating sporocysts, higher percentage of snails shedding cercariae, higher number of cercariae shed and higher mortality in the experimental group that were simultaneously infected with A. vasorum and S. mansoni, when compared to snails infected only with S. mansoni. Snails simultaneously infected with A. vasorum and S. mansoni showed lower hemocyte activation during the first few days of infection, compared to activation induced only by A. vasorum infection. Between 5 and 15 days post-infection (dpi), granulocyte number and nitric oxide (NO) contents of simultaneously infected snails were lower than the S. mansoni-infected snails. Based on the results, we suggest that differences in the level of hemocyte response could explain the increased S. mansoni susceptibility observed in snails simultaneously infected with both parasites. However, when S. mansoni infection occurred after A. vasorum larvae are completely encapsulated, the response against S. mansoni was not altered, and therefore there were no differences in the susceptibility level.

Infecções helmínticas em um rebanho leiteiro na região Campo das Vertentes de Minas Gerais / Helminth infectíon pattern of a dairy herd in Campo das Vertentes region in Minas Gerais state

Bezerros traçadores foram utilizados para avaliar a contaminação sazonal das pastagens por helmintos gastrintestinais e pulmonares em uma propriedade de exploração leiteira na região Campo das Vertentes,Minas Gerais. Os animais se infectaram durante todos os meses do ano. As maiores cargas parasitárias.foram recuperadas no período chuvoso (setembro - abril), e o pico foi observado em abril-maio, final do período chuvoso. As espécies recuperadas foram Cooperia punctata, C. spatulata, Haemonchus contortus, H. similis, Oesophagostomum radiatum, Trichostrongylus axei, T. colubriformis, Trichuris discolor, T. globulosa, Dictyocaulus viviparus e Agriostomum vryburgi. Para avaliar a dinâmica das infecções, foi acompanhado um grupo de vacas com seus bezerros lactantes no período de janeiro de 1999 a fevereiro de 2000. Os valores da contagem de ovos por grama de fezes (OPG) dos bezerros apresentaram dois picos, em maio de 1999 e fevereiro de 2000, enquanto o pico da contagem de OPG das vacas ocorreu em julho e agosto de 2000. Nas coproculturas, os gêneros de maior ocorrência foram Cooperia e Haemonchus nos bezerros, e Haemonchus e Trichostrongylus nas vacas.

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.

Evaluation of the prophylactic effect and curative efficacy of fipronil 1% pour on (Topline) on post-castration scrotal myiasis caused by Cochliomyia hominivorax in cattle.

A field trial was carried out during a summer-fall period on a commercial beef cattle farm in Minas Gerais State, located in the Southeast of Brazil. In order to evaluate the prophylactic effect and the curative efficacy of fipronil in a 1% solution, 200 Zebu crossbred bulls, with ages varying from 20 to 30 months and weights from 233 to 362 kg, were selected. The bulls were assigned by ranked pair to an untreated control group (A) or to a treated group (B), resulting in 100 animals per group. All experimental animals were surgically castrated on day 0, following routine procedures. After castration all animals in the group B were treated with 10 mg/kg bw of a 1% fipronil solution, topically on the dorsal mid-line. The wounds were individually inspected on days: 2, 4, 7, 10, 14, 17, 21, 28 and 35. After castration the animals were naturally exposed to Cochliomyia hominivorax and remained in the same pasture throughout the trial. Among the animals in the control group, 83 were observed to harbor C. hominivorax eggs, with a total of 97 ovipositions, and among those 73 animals had active myiasis. In group B (fipronil 1%), 66 animals showed C. hominivorax eggs, with 92 ovipositions and five animals with active myiasis. Most ovipositions and active myiasis were detected until seven days post-castration for both groups. Wound parasite infestation evidenced bleeding, serous purulent exudation and presence of active C. hominivorax larvae. Treatment with fipronil 1% had a prophylactic effect on scrotal wounds against the development of C. hominivorax larvae in more than 95% of the treated animals for up to 17 days after castration. The treatment showed partial protection of 66% and 50% on days 21 and 28 post-treatment (pt), respectively. Three animals from the control group and one from the treated group showed active screwworms on day 21 pt, and one animal from the treated group and two from the control group also presented C. hominivorax larvae on scrotal wounds on day 28 pt. By the end of the observation period (day 35 pt), the castration wound had healed in all animals. All experimental animals presenting scrotal wounds infested with C. hominivorax larvae were treated with a 1% pour-on formulation of fipronil, on the same day that infestation was observed. Active C. hominivorax larvae were not seen during the monitoring period immediately after treatment. The curative efficacy of fipronil 1% against C. hominivorax larvae infestation in castration wounds was 100%.

Ectopic location of adult worms and first-stage larvae of Angiostrongylus vasorum in an infected dog.

A mongrel dog with angiostrongylosis died suddenly and was submitted to necropsy. Post-mortem findings showed adult worms in ectopic locations. This is the first report of Angiostrongylus vasorum adult worms being found within the pericardial sac and in the lumen of the bladder of an infected dog. A total of 587 A. vasorum first-stage larvae were recovered from different organs and tissues after concentration using Baermann apparatus.

Population dynamics of Lymnaea columella and its natural infection by Fasciola hepatica in the State of Minas Gerais, Brazil.

Studies on the population dynamics and natural infection of Lymnaea columella by Fasciola hepatica were carried out from September 1999 to December 2000 in a low-lying area near Itajubá in the Brazilian State of Minas Gerais. A total of 626 snails were collected monthly at nine different sites, counted, and dissected to search for larvae of F. hepatica. The highest populations of L. columella were reached in October of 1999 and August of 2000, and the highest natural infection rates of snails by F. hepatica were reached in September 1999 (5.2%) and July 2000 (3.9%). The removal by farmers of aquatic plants from the drainage furrows caused a drastic reduction in this snail population.

A method to obtain axenic Angiostrongylus vasorum first-stage larvae from dog feces.

An improved method to obtain a large number of axenic larvae of Angiostrongylus vasorum from fecal samples was developed in the present study. The procedure here in reported consisted of obtaining larvae using a modified Baermann technique, followed by an additional filtration step. This isolation technique recovered almost 90% of the living larvae in a clean preparation. Isolated larvae were submitted to decontamination treatments with either sodium hypochlorite or antibiotic cocktail solutions. The axenic status, as confirmed by oral inoculation of decontaminated larvae into germ-free mice, was only achieved using larvae treated with 0.5% sodium hypochlorite solution for 10 min. The isolation and decontamination treatment did not affect larval viability. Treated larvae remained viable and infective to the invertebrate host.