Experimental Parasitology 121 (2009) 338–341
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Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs Juliana M. Araújo a, Jackson V. Araújo a,*,1, Fabio R. Braga a, Rogério O. Carvalho a, André R. Silva a, Artur K. Campos b a b
Departamento de Veterinária, Universidade Federal de Viçosa, Viçosa (MG) 36570-000, Brazil UNIVIÇOSA, Viçosa (MG) 36570-000, Brazil
a r t i c l e
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Article history: Received 28 March 2008 Received in revised form 3 December 2008 Accepted 15 December 2008 Available online 25 December 2008 Keywords: Nematophagous fungi Duddingtonia flagrans Monacrosporium thaumasium Pochonia chlamydosporia Taenia saginata Biological control
a b s t r a c t The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Helminthosis remains an important problem for public health (Jongwutiwes et al., 2004). In humans, infections by cestodes of the taeniasis/cysticercosis complex affect up to 77 million people. The Taenia saginata has a cosmopolitan distribution and is found widely in most countries that raise cattle. Brazil occupies an important position globally as the largest commercial producer (Alves, 2001). There are no systematic or reliable data available to determine the prevalence of this disease in humans and animals (Pereira et al., 2006). The specialized literature, however, suggests that some groups of people may be at greater risk because of economic, cultural or even religious factors (Pfuetzenreiter and Ávila-Pires, 2000). The occurrence of infection is directly related to poor sanitary conditions and socioeconomic characteristics of the population (González et al., 2002). The life cycle of T. saginata includes a definitive host, an intermediate host and a free-living phase. Adult worms live in the small intestine of humans, the only definitive host. Intermediate hosts of are cattle, where the parasite develops in voluntary muscles (Acha and Szyfres, 1986). Fecal contamination of soil is considered as one of the factors that can favor egg dispersion in the environment (Gemmell and Lawson, 1982). A number of
* Corresponding author. Fax: +55 31 3899 1464. E-mail address:
[email protected] (J.V. Araújo). 1 CNPq scholarship. 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.12.011
preventive measures can be applied to control the spread of infection, but the main strategy consists of interrupting the parasite life cycle (Organizacion Panamericana de la Salud, 1994). Geohelminths are cosmopolitan parasites that depend on multiple factors to stay in the environment and complete their life cycle. Soil containing infective geohelminth eggs is the main source of infection both in humans and animals. Antagonists, such as ovicidal fungi can influence the development and presence of eggs in the soil and cause their destruction (Lysek et al., 1982; Morgan-Jones and Rodriguez-Kabana, 1985). Studies on natural processes of destruction of geohelminth eggs are still in the initial stages, but they represent an alternative approach which, if used with other prophylactic measures, may help control species of epidemiological importance. Nematophagous fungi are divided into predators, endoparasites and opportunists; Pochonia chlamydosporia is ovicidal whereas Monacrosporium thaumasium and Duddingtonia flagrans are predators. Nematophagous fungi are widely distributed and can be found in a great diversity of species as well as in different ecosystems (Gray, 1983). These species have proven effectiveness against eggs and infective larvae of gastrointestinal helminth parasites of domestic animals. (Braga et al., 2008a; Araújo et al., 2007, 2008; Mota et al., 2003). Following a prolonged observational study, Lysek (1976) established a qualitative method for classifying ovicidal activity. This method, first proposed for the activity of Pochonia chlamydosporia against A. lumbricoides eggs, determines that the mechanism of action of an ovicidal fungus is based on three basic
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types of activity and seven subtypes, as follows: (1) physiological; biochemical effect without morphological damage to eggshell; (2) lytic biochemical effect, with progressive morphological, changes of the eggshell and damage to the embryo; and (3) lytic and morphological effect, with hyphal penetration of eggs, attack and death of the embryo; and the subdivisions: (Lysek,1976; Lysek et al., 1982; Lysek and Nigenda, 1989; Lysek and Sterba, 1991). Fungal predators vary in their ability to capture gastrointestinal helminth parasites using traps. The genera Monacrosporium and Duddingtonia stand out in this group (Araújo et al., 2004; Braga et al., 2007). This study aimed at evaluating the ovicidal effect of the nematophagous fungi P. chlamydosporia, M. thaumasium and D. flagrans on eggs of T. saginata, a potential pathogen for humans. 2. Materials and methods 2.1. Organisms Four isolates of nematophagous fungi; two P. chlamydosporia (VC1 and VC4); one D. flagrans (AC001) and one M. thaumasium (NF34) were used in the study. These were originally obtained from soil collected in the municipality of Viçosa, Minas Gerais, Brazil, 20°450 20” latitude and 42°520 40” longitude, using the sprinkling method (Duddington, 1955), modified by (Santos et al., 1991), and kept at the Parasitology Laboratory in the Veterinary Department of the Federal University of Viçosa, MG. 2.2. Taenia saginata eggs T. saginata eggs were morphologically analyzed for integrity under an optical microscope using a 10 objective lens. The eggs were recovered by dissection of adult proglottids obtained from human feces. Proglottid identification followed criteria established by Urquhart et al. (1998) and Bowman et al. (2006). 2.3. Experimental essay The four isolates were kept in test tubes containing 2% corn-mealagar (2% CMA), in the dark, at 4 °C for 10 days. Culture disks, 4 mm in diameter, were removed from grown fungal colonies, plated onto 9cm diameter Petri dishes containing 20 mL of 2% water-agar (2% AA) and stored in the dark, at 25 °C for 10 days. Fungal growth covered the whole of the surface after 10 days. One thousand T. saginata eggs were placed on the surface of 9.0-cm Petri dishes containing 2% AA and the fungal isolates and a control without fungi, with 25 replicates per group. After 5, 10 and 15 days, 100 eggs were removed from the plates treated with P. chlamydosporia (VC1 and VC4), D. flagrans (AC001) and M thaumasium (NF34) and from each control plate (without fungi) as described by Araújo et al. (1995): using a spatula, the eggs were placed on the surface of slides containing a drop of 1% Amam-blue. Eggs were examined under a light microscope (40 objective lens) and scanning electron microscope and evaluated according to Lysek et al. (1982): type 1 effect, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell; type 2 effect, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell, and type 3, lytic effect with morphological changes of embryo and eggshell, with hyphal penetration and internal egg colonization. Data from each time were analyzed by Friedman’s nonparametric test at 5% probability.
(AC001) are shown in Table 1–3. Isolate VC1 showed type 1, 2 and 3 effects at all three times. Isolate VC4 showed slightly less effect at all time points. Examination by optical and scanning electron microscopy showed P. chlamydosporia hyphae on egg surfaces (Fig. 1a and b)
Table 1 Percentage and standard deviation (±) for effects types 1*, 2** and 3*** of the nematophagous fungi Pochonia chlamydosporia (VC1 and VC4), Monacrosporium thaumasium (NF34) and Duddingtonia flagrans (AC001) on Taenia saginata eggs on the 5th day of interaction. Isolates
AC001 NF34 VC1 VC4 Control
Results for type 1, 2 and 3 effects at 5, 10 and 15 days for P. chlamydosporia (VC1 and VC4), M. thaumasium (NF34) and D. flagrans
A
65.0 ± 15.3 72.4A ± 16.0 23.9B ± 6.3 18.4B ± 6.0 0C ± 0
Effect type 2** B
0 ±0 0B ± 0 21.7A ± 17.9 9.9A ± 5.4 0B ± 0
Effect type 3*** 0B ± 0 0B ± 0 12.8A ± 11.6 2.3C ± 1.9 0B ± 0
*
Effect type 1, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell. ** Effect type 2, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell. *** Effect type 3, lytic effect with morphological changes of embryo and eggshell, with hyphal penetration and internal egg colonization. Percentages followed by identical letters (A, B, C) in the same column do not differ (p > 0.05) – Test of Friedman.
Table 2 Percentage and standard deviation (±) for effects types 1*, 2** and 3*** of the nematophagous fungi Pochonia chlamydosporia (VC1 and VC4), Monacrosporium thaumasium (NF34) and Duddingtonia flagrans (AC001) on Taenia saginata eggs on the 10th day of interaction. Isolates
AC 001 NF34 VC 1 VC 4 Control
Effect at 10 days Effect type 1*
Effect type 2**
Effect type 3***
68.1A ± 15.0 74.4A ± 15.9 24.0B ± 8.3 17.5B ± 4.9 0C ± 0
0B ± 0 0B ± 0 24.2A ± 8.5 12.3C ± 5.4 0B ± 0
0B ± 0 0B ± 0 18.2A ± 9.3 7.0C ± 3.8 0B ± 0
*
Effect type 1, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell. Effect type 2, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell. *** Effect type 3, lytic effect with morphological changes of embryo and eggshell, with hyphal penetration and internal egg colonization. Percentages followed by identical letters (A, B, C) in the same column do not differ (p > 0.05) – Test of Friedman. **
Table 3 Percentage and standard deviation (±) for effects types 1*, 2** and 3*** of the nematophagous fungi Pochonia chlamydosporia (VC1 and VC4), Monacrosporium thaumasium (NF34) and Duddingtonia flagrans (AC001) on Taenia saginata eggs on the 15th day of interaction. Isolates
AC001 NF34 VC1 VC4 Control *
Effect at 15 days Effect type 1*
Effect type 2**
Effect type 3***
72.6A ± 16.3 77.3A ± 17.2 22.3B ± 8.4 17.2B ± 5.7 0C ± 0
0B ± 0 0B ± 0 22.2A ± 8.4 17.2C ± 6.3 0B ± 0
0B ± 0 0B ± 0 9.7A ± 5.7 8.0A ± 4.0 0B ± 0
Effect type 1, physiological and biochemical effect without morphological damage to eggshell, with visualization of hyphae adhered to eggshell. Effect type 2, lytic effect with morphological changes in embryo and eggshell, without hyphal penetration through the eggshell. *** Effect type 3, lytic effect with morphological changes of embryo and eggshell, with hyphal penetration and internal egg colonization. Percentages followed by identical letters (A, B, C) in the same column do not differ (p > 0.05) – Test of Friedman. **
3. Results and discussion
Effect at five day Effect type 1*
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Fig. 1. Pochonia chlamydosporia hyphae and chlamydospore (white arrow) on the surface and inside of Taenia saginata eggs (black arrow) on the 5th day of interaction. Optical Microscopy – 40 objective lens and Scanning Electron Microscopy. (Bar): 10 lm.
Fig. 2. Pochonia chlamydosporia hyphae and chlamydospores (white arrow) in the inside of Taenia saginata eggs (black arrow). Optical Microscopy – 40 objective lens.
Fig. 3. Pochonia chlamydosporia hyphae (white arrow) and destroyed Taenia saginata eggs (black arrow). Optical microscopy – 40 objective lens.
at 5 days, hyphae and chlamydospores presence inside the eggs (Fig. 2a and b) at 10 days, as well as destroyed eggshells (Fig. 3a and b) at 10 and 15 days of interaction, characterizing type 3 effects (Lysek, 1976). Fig. 4a and b show T. saginata eggs of the control treatment, without fungus, at 10 and 15 days. No significant differences were found between the isolates VC1 and VC4 for type 1 effect (P > 0.05). VC1 was more active than VC4 for type 2 and 3 effects, which was significant (P < 0.05) at 10 and 15 days for a type 2 effect, and at 5 and 10 days for a type 3 effect. In the current study, isolate VC1 was more effective mainly for a type 3 effect, which characterizes the fungus as potentially ovicidal (Lysek, 1976). In a similar study, Braga et al. (2008a) evaluated the effectiveness of VC1 and VC4 isolates on Fasciola hepatica eggs and found no significant difference (P > 0.01) in the results for type 3 effect. The same authors also evaluated the effect on Schistosoma mansoni eggs and found no difference (P > 0.01) for type 3 effect at the studied intervals (Braga et al., 2008b). However, when the 5, 10 and 15 day intervals used here are compared with the intervals of 7, 14 and 21 days studied by Braga et al. (2008a,b), the ovicidal action of P. chlamydosporia (VC1 and VC4) was very similar,
Fig. 4. Show T. saginata eggs of the control treatment, without fungus, at 10 and 15 days. (Bar): 10 lm.
demonstrating that the fungus is effective at any interval. The M. thaumasium (NF34) and D. flagrans (AC001) isolates only showed for a type 1 effect at 5, 10 and 15 days. The effect of M. thaumasium (NF34) and D. flagrans (AC001) on Ascaris lumbricoides eggs were studied at intervals of 7, 10 and 14 days and only showed type 1 effect with the activity of isolate NF34 being higher than AC001 only on the 7th day (Braga et al., 2007). Araújo et al. (2007) reported significant effectiveness of M. thaumasium (p < 0.05) on gastrointestinal nematodes in goats in semiarid northeastern Brazil. In another study, Araújo et al. (2006) tested the capacity of D. flagrans to pass through the gastrointestinal tract of goats, finding significant reduction (P < 0.05) in the number of recovered Strongyloides pappillosus and Haemonchus contortus infective larvae. In the present work, these fungi only showed hyphae adhered to the eggshell of T. saginata, without egg destruction, and are therefore not classified as ovicidal (Lysek, 1976). T. saginata eggs are very resistant in the environment because of the protective outer shell (embryophore) (Silva, 2005). T. saginata eggs may be viable over periods of approximately 4 to 12 months and are also resistant to conventional sewage treatment (Huggins, 1989; Reiff, 1994). Braga et al. (2008a) found that P. chlamydosporia showed in vitro activity against F. hepatica eggs, but this activity was low since they are also resistant in the environment (Burger and Stoye, 1978). Therefore, the ovicidal action of P. chlamydosporia on eggs with thicker shell may be low, as the results of T. saginata and F. hepatica showed. Although the penetration mechanism of ovicidal fungi into parasitized eggs is still not totally clear, enzymatic activity is believed to be the main process for attack and penetration of eggs (Lysek and Sterba, 1991). Lysek and Nigenda (1989) proposed that, once the methodology for ovicidal activity has been improved, one should focus on the comparison of ovicidal capacity of different species. These authors also mentioned the importance of these studies for the decontamination of soil from helminths, since ovicidal nematophagous fungi are natural organisms already present in the environment. In this context, alternative measures such as the biological control by natural antagonistic fungi of gastrointestinal helminth parasites that propagate in fecal environment are very important, (Mota et al., 2003; Araújo et al., 2004). Acknowledgment This work was financially supported by the CNPq. References Acha, P., Szyfres, B., 1986. Zoonosis y enfermidades trasmissible comunes al hombre y a los animales. Organizacion Panamericana de La Salud, Washington (in Spanish). Alves, D.A., 2001. As dificuldades na inspeção de frigoríficos brasileiros no mercado internacional: um estudo sobre a comercialização da carne bovina in natura. Revista Nacional da carne 25, 96–114 (in Portuguese). Araújo, J.V., Braga, F.R., Araújo, J.M., Silva, A.R., Tavela, A.O., 2008. In vitro evaluation of the effect of the nematophagous fungi Duddingtonia flagrans, Monacrosporium
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