Elucidating the life cycle of opossum parasites: DNA sequences reveal the involvement of planorbid snails as intermediate hosts of Rhopalias spp. (Trematoda: Echinostomatidae) in Brazil.

Echinostomatid digeneans belonging to the genus Rhopalias are intestinal trematodes found mainly in opossums in the New World. The genus comprises seven species, but their life cycles and intermediate hosts have been unknown until now. During our long-term study carried out in freshwater habitats within the state of Minas Gerais, Southeast Brazil, echinostomatid cercariae lacking collar spines were found in planorbid snails Biomphalaria glabrata, Biomphalaria straminea, Drepanotrema lucidum and Gundlachia ticaga in six different batches of snail samples collected between 2010 and 2019. Morphologically, the larvae reported herein are morphologically consistent with each other and characterized by the presence of 2-3 large ovoid or spherical corpuscles in each main duct of the excretory system, resembling to Cercaria macrogranulosa previously described from the same region of Brazil. Partial sequences of the ITS (ITS1-5.8S-ITS2) region and 28S gene of the nuclear ribosomal RNA operon, and partial sequences of mitochondrial nad1 and cox1 genes were obtained and compared with the data available for members of the family Echinostomatidae. Nuclear markers indicate that all samples of cercariae evaluated in the present study can be assigned to Rhopalias, but distinct from North American isolates of Rhopalias macracanthus, Rhopalias coronatus and Rhopalias oochi (divergence 0.2-1.2% in 28S and 0.8-4.7% in ITS). The lack of differences verified in both 28S and ITS in 5 out 6 studied samples suggested that they belong to the same species. However, nad1 sequences revealed that our cercariae correspond to three distinct species of Rhopalias (interspecific divergence: 7.7-9.9%), named here as Rhopalias sp. 1, found in B. straminea and G. ticaga, Rhopalias sp. 2 found in B. glabrata and D. lucidum, and Rhopalias sp. 3 also found in D. lucidum. They also differ by 10.8-17.2% from a North American isolate of R. macracanthus sequenced in this study. The cox1 sequences obtained for Rhopalias sp. 1 and Rhopalias sp. 2 (but not Rhopalias sp. 3) reveal that they are distinct from North American isolates of R. macracanthus (genetic divergence 16.3-16.5% and 15.6-15.7%, respectively), R. coronatus (9.2-9.3% and 9.3-9.5%) and Rhopalias oochi (9.0% and 9.5-10.1%). Encysted metacercariae with general morphology similar to that of the body of cercariae were found in tadpoles of Rhinella sp. from the same stream where snails harbored Rhopalias sp. 2, suggesting that the amphibians could act as second intermediate hosts of species of Rhopalias. Data obtained provide the first insights into the life cycle of this unusual echinostomatid genus.

Germination capacity of the Pochonia chlamydosporia fungus after its passage through the gastrointestinal tract of domestic chickens (Gallus gallus domesticus).

This study evaluated the germination capacity of Pochonia chlamydosporia (VC4) fungus after its passage through the gastrointestinal tract of domestic chickens and its interaction with Ascaridia galli and Heterakis gallinarum eggs. Twenty-two domestic chickens were divided in two groups: control group (G1) received shredded corn substrate without VC4; and treatment group (G2) received a single dose of 29 g corn substrate containing 3.3 × 106 conidia/chlamydospores (VC4). Subsequently, chicken fecal samples were collected at intervals of 0, 6, 8, 10, 12, 18 and 24 h. Petri dishes from fecal samples of the treated group (G2) were subdivided (G2a and G2b), and then replicated in 2% agar-water medium for the microbiological test. After VC4 growth, approximately 200 eggs of A. galli (G2a) and H. gallinarum (G2b) were added to each subgroup to evaluation of ovicidal activity. There was fungal viability after passage through chicken gastrointestinal tract and egg predation of 59.9% and 43.2% for A. galli and H. gallinarum, respectively. The present work demonstrates the ability of the fungus P. chlamydosporia to survive after passing through the gastrointestinal tract of domestic chickens, an extreme environment (low pH, enzymes, microbiota and mechanical action), and still germinate after being excreted with feces.

Duddingtonia flagrans formulated in rice bran in the control of Oesophagostomum spp. intestinal parasite of swine.

Three experimental assays with Duddingtonia flagrans (isolated AC001) were carried out. The growth of the genus Duddingtonia present in formulation of rice bran, its predatory capability on Oesophagostomum spp. infective larvae (L3) in petri dishes (assay 1), its action in faecal cultures with eggs of that parasite (assay 2) and isolate's capability of predation after passing through gastrointestinal tract of swine (assay 3) was evaluated. At assay 3, feces were collected at time intervals of 12, 24, 36, 48, and 60 h after feed animals with the formulation. Assays 1 and 2 showed a statistical difference (p < 0.01) by the F test when comparing the treated group with the control group. At the both assays, was observed in the treated group a reduction percentage of 74.18% and 88.38%, respectively. In assay 3, there was a statistical difference between the treated group and the control group at all collection times (p < 0.01). Regarding the collection periods, there was no statistical difference over time in the treatment group (p > 0.05). The results demonstrate that the fungal isolate AC001 formulated in rice bran can prey on L3 of Oesophagostomum spp., in vitro and after passing through the gastrointestinal tract, without loss of viability. This isolate may be an alternative in the control of Oesophagostomum spp. in swine.