New approaches to studying morphological details of intramolluscan stages of Angiostrongylus vasorum.

Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-ß-tubulin, anti-α-tubulin, anti- α-actin, anti-ß-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.

Identification of a phenoloxidase- and melanin-dependent defence mechanism in Achatina fulica infected with Angiostrongylus vasorum.

BACKGROUND: Angiostrongylus vasorum has different freshwater aquatic and terrestrial gastropod molluscs as an intermediate host, e.g. Arion spp. The mollusc Achatina fulica is a danger to public health, given the large diversity of nematodes utilizing it as an intermediate host, such as the parasites of the genus Angiostrongylus, of importance in human and veterinary medicine. Achatina fulica has been shown to have an excellent capacity for maintaining outbreaks and natural infections with A. cantonensis in Asia. Within the mollusc, the nematode parasites activate haemocytes and/or haemolymph factors and in some invertebrates, phenoloxidase (PO), that induces the release of toxic elements and eliminates the parasites. Despite the importance of A. fulica in the life-cycle of nematodes, little is known regarding the defence mechanisms involving PO in molluscs infected with nematodes. Here, the presence of PO and nitric oxide (NO) in the haemolymph and haemocytes of A. fulica infected with first-stage (L1) larvae of Angiostrongylus vasorum was evaluated, together with the presence of melanin in the cephalopod mollusc tissue. RESULTS: An increase in PO at one day post infection (dpi), in comparison with the control using the substrates L-tyrosine (F(4,90) = 6.73, P = 0.00006), L-DOPA (F(4,90) = 22.67, P = 0.02) and p-phenylenediamine (PPD) (F(4,90) = 27.58, P = 0.0019), was observed. PO increase coincided with the presence of melanin in the cephalopodal tissue. At 8 dpi, PO activity, compared to L-DOPA (F(4,90) = 22.67, P = 0.00002) and PPD (F(4,90) = 27.58, P = 0.079) decreased, while melanin increased. At 13 dpi, PO decreased with PPD (F(4,90) = 27.58, P = 0.000015) and also the amount of melanin observed in histology. At 30 dpi, PO increased along with the substrates L-DOPA and PPD, while melanin decreased. NO levels increased until 8 dpi, and decreased after 13 dpi. CONCLUSIONS: To our knowledge, this is the first study that illustrates PO activity in a helminth-infected A. fulica and provides the first observation of an L-tyrosine dependent PO activity in molluscs infected with A. vasorum. This work suggests that PO pathway may help to control A. vasorum infection in A. fulica.

Dynamics of haemocytes from Pseudosuccinea columella circulating infected by Fasciola hepatica / Dinâmica de hemócitos circulantes de Pseudosuccinea columella infectados por Fasciola hepatica

Abstract The lymnaeids are important in the epidemiology of Fasciola hepatica, a neglected and endemic zoonosis. The interaction between the internal defense system of Pseudosuccinea columella and F. hepatica has been little studied. In the present study the effect of infection by F. hepatica on P. columella circulating haemocytes was investigated. Changes in the average number of total circulating haemocytes have been observed at 30 minutes post-infection and 1, 7, 10, 14, 21, 28 and 50 days post-infection (dpi). Miracidia were observed head-foot and mantle at 30 minutes post-infection. Miracidia/Sporocysts in the mantle skirt 1 dpi, and fully formed sporocysts were observed in the head-foot at 7 dpi. Rediae became evident at 10 dpi and were located between the haemocoel and the muscles from 14 dpi; 50 dpi, the rediae in the digestive gland contained cercariae. The statistical analysis of the total haemocytes of P. columella infected by F. hepatica showed significant differences on the 30 minutes post-infection and 1, 14, 21, and 28 dpi in comparison to uninfected molluscs (0 dpi). Therefore, the interference observed on the internal defence system of P. columella may have direct association with the development of F. hepatica.

Resumo Os limnaeideos são importantes na epidemiologia de Fasciola hepatica, uma zoonose negligenciada e endêmica. A interação entre o sistema interno de defesa de Pseudosuccinea columella e F. hepatica tem sido pouco estudada. No presente estudo, investigou-se o efeito da infecção por F. hepatica nos hemócitos circulantes de P. columella. Alterações no número médio de hemócitos circulantes foram observadas aos 30 minutos e 1, 7, 10, 14, 21, 28 e 50 dias após a infecção (dpi). Miracídios foram observados na região cefalopodal e manto aos 30 minutos após a infecção. Miracídio/esporocistos foram observados no colar do manto ao 1 dpi, e esporocistos totalmente formados na região cefalopodal aos 7 dpi. Rédias tornam-se evidentes aos 10 dpi entre a hemocele e músculos a partir de 14 dpi; e rédias com cercárias próximas a glândula digestiva aos 50 dpi. A análise estatística dos hemócitos totais de P. columella infectados por F. hepatica demonstrou diferenças significativas nos 30 minutos pós-infecção e 1, 14, 21 e 28 dpi em comparação aos moluscos não infectados (0 dpi). Portanto, a interferência observada no sistema de defesa interna de P. columella pode ter associação direta com o desenvolvimento de F. hepatica.

Dynamics of haemocytes from Pseudosuccinea columella circulating infected by Fasciola hepatica.

The lymnaeids are important in the epidemiology of Fasciola hepatica, a neglected and endemic zoonosis. The interaction between the internal defense system of Pseudosuccinea columella and F. hepatica has been little studied. In the present study the effect of infection by F. hepatica on P. columella circulating haemocytes was investigated. Changes in the average number of total circulating haemocytes have been observed at 30 minutes post-infection and 1, 7, 10, 14, 21, 28 and 50 days post-infection (dpi). Miracidia were observed head-foot and mantle at 30 minutes post-infection. Miracidia/Sporocysts in the mantle skirt 1 dpi, and fully formed sporocysts were observed in the head-foot at 7 dpi. Rediae became evident at 10 dpi and were located between the haemocoel and the muscles from 14 dpi; 50 dpi, the rediae in the digestive gland contained cercariae. The statistical analysis of the total haemocytes of P. columella infected by F. hepatica showed significant differences on the 30 minutes post-infection and 1, 14, 21, and 28 dpi in comparison to uninfected molluscs (0 dpi). Therefore, the interference observed on the internal defence system of P. columella may have direct association with the development of F. hepatica.

Evaluation of techniques for recovery of Angiostrongylus vasorum from Achatina fulica, a potential intermediate host

Mollusks are intermediate hosts of Angiostrongylus vasorum, which is a parasite with zoonotic potential for which canids are the definitive host. In this study, 180 specimens of Achatina fulica that had been experimentally infected with 1,000 L1 of A. vasorum were finely chopped to evaluate four tissue digestion techniques: 1% potassium hydroxide (group I); 3% hydrochloric acid (group II); 1% hydrochloric acid and pepsin (group III) with procedures at 37 ºC in a double boiler; and Baermann at 42 °C (group IV). On the first, eighth and thirtieth days after infection, L1, L2 and L3 were obtained, and 240 L3 recovered from group III were offered to a dog. The results were the following: group I: 59% L1, 7% L2, 13% L3; group II: 31% L1, 13% L2, 27% L3; group III: 23% L1, 22% L2, 30% L3; and group IV: 0.3% L1, 0.02% L2, 5% L3. Live larvae wereobserved in group III: 15.5% L1, 8.22% L3; group II: 9.8% L3; and group IV: 100% L1, L2 and L3. Larvae were detected 56 days after the dog became infected. All larvae stages of A. vasorum were recovered and identified through the techniques evaluated, thus confirming the potential of A. fulica as an intermediate host of the nematode