Toxocariasis is the clinical presentation of human infection by Toxocara spp., a roundworm that lives in the small intestines of domestic dogs and cats. The infection occurs by the accidental ingestion of embryonated Toxocara eggs present in contaminated soil or on dirty hands6,14. Toxocariasis is present worldwide and is a consequence of the human habit of keeping dogs and cats for company, which favors the persistence of the parasite in the environment and its transmission13,22. Despite its extensive geographical distribution, infection is more frequent in tropical and subtropical regions, especially in populations with poor sanitary conditions20. Human T. canis infection is a public health concern in the Americas, Europe and in all developing countries. However, a full appreciation of the global burden of this disease may be greatly underestimated18.

Toxocara spp. eggs, as the eggs of other geohelminths, have a high degree of resistance to adverse environmental conditions and diverse chemicals, since they are protected by a thick, complex shell. This shell is made up of three membranes or layers – the outer vitelline layer, a middle chitinous layer, and the inner lipidic layer – that makes eggs resistant to chemicals and temperature changes, allowing them to survive outside the host for long periods22. Consequently, soil contamination with infective eggs is a worldwide health issue, and that is why the interest in finding biological control agents to reduce this contamination has increased in the past decades4.

Fungal parasitism on nematode eggs is a natural biological phenomenon that can be used for biological control of geohelminth eggs in the environment, since they are the most resistant stage in the life cycle of nematodes4,5. The penetration process of the hypha through the egg shell has not been completely elucidated yet. In 1995, Bonants et al.8 were the first to mention that the fungi colonization mechanism may be mechanical and/or enzymatic. Special penetration organs (“appresoria”) formed from the hypha help the fungus apply pressure on the egg shell (mechanical effect). Other investigations suggest the involvement of exoenzymes such as proteases and chitinases breaking up egg shells (enzymatic mechanism)4.

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by the different fungal species. Thus, a saprophytic fungus may exert ovicidal or ovistatic effects, where the ovistatic ability is shown by the delay in embryo development or inhibition with no morphological damage to the egg shell12. Therefore, several researchers have assayed the in vitro effect of different fungi on geohelminth eggs. Knowledge about the effect of the genus Chrysosporium is scarce, since there is no register of studies on the subject except for the study carried out by Ciarmela et al.10, who characterized Chrysosporium merdarium species as having very high ovicidal activity on Toxocara canis eggs, along with other soil saprophytic fungi.

The purpose of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium, Chrysosporium indicum and Chrysosporium keratinophylum, on T. canis eggs.

The effect of fungi on the eggs was evaluated according to the alterations observed in the surface and the changes in the normal characteristics of the eggs. Figure 1A and B shows normally developed T. canis eggs of the control group after 14 days of incubation, with no morphological alterations and intact shells, exhibiting their typical surface.

Figure 1.

SEM observation of T. canis eggs of the control group, incubated for 14 days. (A) Structure of the shell and shape of the egg preserved without any alterations (1100×). (B) Magnification of the egg shell (8000×).

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In contrast to the control group, T. canis eggs submitted to the interaction assays with both Chrysosporium were observed submerged into a hyphal network as from the 7th day of incubation and penetration (“appresoria”) organs that finally managed to penetrate the shell appeared. Figures 2 and 3B show hyphae surrounding the egg. Appresoria penetrating the shell can be observed in Figure 3A and B. hyphae around the eggs make the shell softer and thinner. Changes in the typical egg membrane can be observed in Figures 2 and 4.

Figure 2.

SEM observation of T. canis eggs + C. keratinophylum incubated for 14 days. Alterations in the shell structure (black arrow), presence of mycelia inside the egg and egg rupture (white arrow) (1400×). Type 3 effect.

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Figure 3.

SEM observations of T. canis eggs + C. keratinophylum, incubated for 7 days. (A, B) Appresoria structures (white arrow) (2500×) and the network of mycelia formed by hyphae around the egg (black arrow) (B)

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Figure 4.

SEM observations of T. canis eggs + C. indicum incubated for 14 days. Egg deformation due to “consumption or utilization” of the embryo (black arrow) and alterations in the shell structure (white arrow) (500×).

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The highest percentage of affected eggs was observed between days 7 and 14. After 7 days of incubation, 46% and 69% of the eggs were altered by C. indicum and C. keratinophylum, respectively. On day 14, this percentage had increased 68.7% with C. indicum and 74% C. keratinophylum. From day 14 onwards, no major changes were detected.

Non-significant differences were obtained between both Chrysosporium species.

Several structural changes of the egg shell occur when fungal hyphae contact the egg, consequently affecting the embryo. This observation of hyphae inside the egg can be possible only if, when assembled for the SEM, the shell breaks and allows to see what happens inside (Fig. 2), otherwise these observations are not possible by this technique.

According to Lysek and Sterba19, the effect of the fungi classified as Type 3 prevails in our study. This effect includes hypha penetration into the eggs and alterations occurring both in the shell and the embryo.

In order to contribute to the biological control of geohelminths, many studies in Latin America have assessed the effect of different fungi on T. canis eggs in Argentina3,10,11,16,17 and Brazil1,2,9,12,15. In Europe, the contribution of Mazurkiewicz-Zapalowicz et al.21 regarding the effects of several species of soil saprophytic fungi on T. canis deserves to be mentioned.

Chrysosporium is a filamentous keratinophilic fungus, commonly isolated from soils, vegetal material, manure and birds. It lives among the remains of hair and feathers on the soil. Besides being a common contaminant, it is occasionally isolated from human infections. This genus adapts to hot weather areas, is constant and dominant throughout northern Argentina and is cosmopolitan as regards its distribution23.

The use of SEM allowed us to observe clearly the interaction fungi-eggs and how C. indicum and C. keratinophylum affect the normal development and alter the structure of T. canis eggs. We also noted that these alterations increased depending on the length of exposure. A similar action was reported for only one Chrysosporium species by Ciarmela et al.10 in their studies on C. merdarium. These authors report a high in vitro ovicidal activity of C. merdarium and describe that the fungus growth and the egg alteration was visible after 21 days of incubation. Unlike our results, we already observed an active interaction as from the 7th day. At this time, a hyphal network surrounding the eggs with appressorium formation and the thinning and smoothness of the shell maintained throughout the days of exposure were observed with both Chrysosporium. As it was documented for C. merdarium, we observed that T. canis eggs had smooth shells since the 14th day post-incubation10.

It is worth considering that the eggs used in this study to conduct the in vitro test were immature or unembryonated. Lysek and Sterba19 mentioned other investigations in which Paecilomyces lilacinus (current name Purpureocillium lilacinum) colonizes Globodera pallida eggs more readily when they are in the early stages of development. Depending on their maturity, these authors reported that eggs have different degrees of resistance to being invaded. For most fungi, when eggs are in a more advanced stage or there is larval development inside them, the ovicidal activity and the ability to colonize eggs are reduced19.

Due to their ovicidal nature, P. lilacinum and Pochonia chlamydosporia are the most studied fungi in vitro. Only P. lilacinum has been isolated from soils in northeastern Argentina23, but its in vitro activity has been shown widely enough, and therefore was not considered for this study. P. lilacinus has been studied by Basualdo et al.3, who proved that T. canis eggs colonized by this fungus do not fully develop and the fungus produces special penetration organs, causing mechanical damage to the egg. Likewise, it is important to observe that both species of Chrysosporium tested in this study have formed hyphal networks around the eggs with attack-specialized mycelium (appresoria), which favored the penetration of this ovicidal fungus through resistant egg-shells inside the eggs, with the same effect.

Carvalho et al.9 also studied the interaction of P. lilacinum and P. chlamydosporia on T. canis eggs. These researchers believe that both fungi can destroy the eggs under laboratory conditions, and claim that the longer the contact between fungus and egg, the more efficient the ovicidal activity will be. In our study, SEM observations allowed us to reach similar conclusions on the effect of C. indicum and C. keratinophylum, with a high activity of these fungi from 7th day, being able to alter and penetrate the eggs from the 14th day.

Despite their slow growth, Ciarmela et al.10 show, their mechanically and enzymatically-driven ovicidal activity for the species C. merdarium, since, by the 14th day post-incubation, eggs with a smooth shell were observed, similarly to what we have reported in our assay (Fig. 2). Despite their high antagonic activity, Ciarmela considers it a control agent only for limited use because it may affect human beings10; however, the presence of these Chrysosporium species in soils can play a very important role as natural biological control agents.

This is the first assay conducted in northeast Argentina. We can conclude that both C. indicum and C. keratinophylum have, in vitro, a high capacity to destroy T. canis eggs, which can be well observed and described under SEM. We should deepen our studies to complete the knowledge about the mechanisms used by these fungi to, fully or partially, destroy T. canis eggs, as well as, to learn about the influence of the type of soil, humidity, temperature, and presence of other organisms on the in vivo action of these fungi on geohelminth eggs.

The authors declare no conflict of interest.