Predatory activity of chlamydospores of the fungus Pochonia chlamydosporia on Toxocara canis eggs under laboratory conditions

The objective of this study was to use chlamydospores of the fungus Pochonia chlamydosporia (isolates VC1 and VC4) against Toxocara canis eggs in a 15-day in vitro assay. One thousand T. canis eggs were placed in Petri dishes containing 2% water agar medium with different concentrations of chlamydospores (1,000, 10,000 or 100,000) of each fungal isolate of P. chlamydosporia (treated groups) and 1,000 eggs in Petri dishes without fungus (control group). Egg counts were performed to determine the ovicidal activity, which was classified as three effect levels: type 1, type 2 and type 3. Significant differences (P < 0.01) in egg destruction were found in comparison with the control group. The highest percentage of egg destruction was found in plates containing 100,000 chlamydospores (68.5% for VC1 and 70.5% for VC4). Chlamydospores of P. chlamydosporia were effective in destroying T. canis eggs and may contribute in the future towards combating the eggs of this parasite.

Toxocara canis is the causative agent of diseases known as visceral larva migrans and ocular larva migrans in humans (REY, 2008). In addition, close association between humans and domestic animals is considered to be a public health hazard (VASCONCELLOS et al., 2006). Increasing numbers of pets have led to greater contact with humans, thereby increasing the risk of exposure to zoonosis (GENNARI et al., 1999). These authors also reported that children, especially at preschool ages, are commonly infected through ingestion of embryonated eggs.
Dogs acquire T. canis by ingesting eggs that contain L 3 or by preying upon rodents, reptiles and birds that may be infected, and act as paratenic hosts. Transplacental and lactogenic migration also account for infection (ALDAWEK et al., 2002;LEITE et al., 2004;MONTEIRO, 2010;BOWMAN, 2010). Use of anthelmintic drugs is a common approach towards controlling adult parasites in hosts. However, resistance to some drugs used for controlling infection in host animals, especially pyrantel, has been mentioned in the literature (KOPP et al., 2008). On the other hand, there are no reports relating to controlling infective forms (larvae and eggs) in the environment (REY, 2008).
Nematophagous fungi have been studied under laboratory conditions as an alternative for controlling T. canis eggs and other potentially zoonotic helminths (ARAÚJO et al., 2009;CARVALHO et al., 2010). Pochonia is a chlamydospore-producing genus capable of withstanding such conditions. However, there are only a few studies on use of chlamydospore structures for destruction of helminth eggs in vitro (BRAGA et al., 2011). These have demonstrated that species of the genus Pochonia are inoffensive to animals and humans and are able to destroy helminth eggs in a short time using special pressure organs called appresoria.
The aim of this study was to assess the in vitro ovicidal activity of different concentrations of chlamydospores from different fungal isolates of Pochonia chlamydosporia on T. canis eggs.
T. canis eggs were dissected from adult specimens expelled from naturally infected dogs and morphologically analyzed by means of optical microscopy (10× objective), as described by Urquhart et al. (1998). The eggs were washed in distilled water, centrifuged (1,000 g for 5 minutes) and incubated at 25 °C for 14 days in a solution containing 0.005% streptomycin sulfate and 0.01% chloramphenicol. This procedure was used to obtain embryonated eggs. This assay was based on the protocol described by Araujo et al. (1995).
Chlamydospores were obtained from two P. chlamydosporia isolates (VC1 and VC4). This fungus has been maintained under continuous culture at the Parasitology Laboratory of the Department of Veterinary Medicine, Federal University of Viçosa, Minas Gerais, Brazil. The fungal isolates were grown in Petri dishes containing 20 mL of YPSSA medium (4 g of yeast extract; 1 g of K 2 HPO 4 ; 0.5 g of MgSO 4 ; 20 g of soluble starch; 20 g of agar; and water to complete 1 liter of solution) at 25 °C in the dark for 10 days. The dish surfaces were rinsed (10 mL of distilled water) using a paintbrush. The suspension obtained was sieved into a plastic container to remove mycelium fragments. Chlamydospores were recovered and quantified using a Neubauer chamber, following the identification methods proposed by Gams and Zare (2001).
The following protocol established by Braga et al. (2011) was used. The experiment comprised six treatments consisting of 1,000 eggs of T. canis plated on 9 cm Petri dishes containing 2% water agar (2% WA) medium with the different concentrations of chlamydospores (1,000, 10,000 and 100,000) from each fungal isolate (VC1 or VC4). A control group containing 2% WA, consisting of 1,000 eggs of T. canis, was established for each concentration of chlamydospores, with six repetitions.
At the end of the experimental period (15 days), 100 eggs of T. canis were removed from each dish (treated and control dishes) and examined for ovicidal activity (LÝSEK et al., 1982): type 1, lytic effect without morphological damage to eggshell, with hyphae adhering to the shell; type 2, lytic effect with morphological alteration of embryo and eggshell, without hyphal penetration through the eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, as well as hyphal penetration and internal colonization. Eggs were collected from each plate containing the isolate and from the control without fungus, as described by Araújo et al. (1995), placed on glass slides with a drop of 1% Amam blue and evaluated under a 40x lens. The data were analyzed by means of the Friedman nonparametric test at the 1% probability level (AYRES et al., 2003).
Pochonia chlamydosporia chlamydospores were effective in destroying T. canis eggs. There was a significant difference (P < 0.01) in egg destruction at the end of the experiment between the treatment and control groups (Figure 1a-f ).
For the concentrations of 1,000 chlamydospores, the percentages of egg destruction were 48.0% (VC1) and 50.0% (VC4); for the concentrations of 10,000 chlamydospores, the percentages of egg destruction were 66.0% (VC1) and 69.0% (VC4). The highest percentages of egg destruction were found with the concentration of 100,000 chlamydospores of both isolates (68.5% for VC1 and 70.5% for VC4) at the end of 15 days (Table 1). Table 1. Percentages and standard deviations for type 1, 2 and 3 effects of ovicidal activity against Toxocara canis eggs, caused by P. chlamydosporia (isolates VC1 and VC4 ) at concentrations of 1000, 10000 and 100000 chlamydospores and control group in 2% water agar (2% WA), after 15 days of interaction. The chlamydospores of the isolates VC1 and VC4 used at the three concentrations (1,000, 10,000 and 100,000) destroyed parasite eggs effectively. Use of chlamydospores against gastrointestinal helminth parasites has been reported for many years with the positive result of effective destruction (LARSEN, 1999). There is, however, a lack of studies concerning the ovicidal activity of chlamydospores under controlled conditions (BRAGA et al., 2011). Use of nematophagous fungi as biological control agents is promising, although contamination during chlamydospore production is still a serious obstacle (ARAÚJO et al., 2004).

Chlamydospores
Comparing the results found here with those reported by Braga et al. (2011), it can be affirmed that: (1) both studies used increasing concentrations of chlamydospores that effectively destroyed eggs in vitro; (2) in the present study, eggs of an ascarid parasite were used and were morphologically different from the eggs of Cestoda used by Braga et al. (2011), which provides justification for further studies to identify the possible obstacles to use of P. chlamydosporia, given that geohelminths are present in large quantities in the environment; (3) the 2% WA medium used in the in vitro tests of both studies showed that P. chlamydosporia has low nutritional requirements, although, as Costa et al. (2001) pointed out, this does not reflect the natural environmental conditions; and finally, (4) we confirmed that the ovicidal activity of P. chlamydosporia continues over long time periods, as reported by Braga et al. (2011). This last point is an important finding, because helminth eggs generally have completely different developmental stages. In the present study, the decision to use only one time interval (15 days) was based on the prepatent period of 15 to 21 days for T. canis. The results suggest that the P. chlamydosporia isolates VC1 and VC4 can be used for biological control of ascarid eggs.
T. canis eggs were most effectively destroyed at the highest concentration of chlamydospores. This result was already expected, since Braga et al. (2011) also reported that eggs of Taenia taeniaeformis were most effectively destroyed at the concentration of 20,000 chlamydospores. On the other hand, in a richer culture medium such as 2% cornmeal agar (BRAGA et al., 2011), chlamydospore production may be better observed, thus perhaps suggesting that there is greater ovicidal activity.
Several reports have shown the efficacy of the isolates VC1 and VC4, with no difference in egg destruction. There are, however, few studies on the effectiveness of chlamydospores from these fungal isolates. Carvalho et al. (2010) showed that P. chlamydosporia isolates grown in 2% WA were effective in destroying T. canis eggs using time intervals of 7, 14 and 21 days, with ovicidal activity of 20.3% (VC1) and 21.7% (VC4) at the end of the experiment. Frassy et al. (2010) used the same isolates of P. chlamydosporia fungus on T. canis eggs and registered percentage values for type 3 effect of 43.3% and 47.3%, respectively, after 15 days.
It should be noted that the above studies were conducted using fungal isolates grown in Petri dishes containing the 2% WA medium. However, in the present work, the fungal isolates (VC1 and VC4) were evaluated in the form of chlamydospores, which are resistant vegetative structures. These germinated on the plates and destroyed T. canis eggs. Hence, it can be suggested that use of chlamydospores of P. chlamydosporia fungus might present better results with regard to biological control of this nematode. This is the first report on using different concentrations of chlamydospores of P. chlamydosporia for destruction of T. canis eggs. All the concentrations of chlamydospores of the isolates VC1 and VC4 were efficient in destroying the eggs, but further studies are required for future use of this fungus for biological control of T. canis eggs.