Changes in the viability of Strongyloides ransomi larvae ( Nematoda , Rhabditida ) under the influence of synthetic flavourings

One of the most common nematodes of pigs globally is Strongyloides ransomi Schwartz and Alicata 1930. It usually causes aggravation of physiological indicators of its hosts and damage to their immune system. Also it is a good modelling object for the evaluation of the antiparasitic activity of new antihelminthic drugs. We conducted laboratory experiments to assess the effect of flavouring additives with flower odour (benzaldehyde, citral, D-limonene and β-ionone) upon the viability of S. ransomi larvae. The mortality rate was calculated for 24 hours exposure at four concentrations of each substance (10, 1, 0.1 и 0.01 g/l) with eight replications. The lowest LD50 values were obtained for citral (97 mg/l) and benzaldehyde (142 mg/l). These substances are recommended for further evaluation of their antihelminthic effect in experiments using laboratory animals. Unlike other substances, the effect of β-ionone and D-limonene even at a concentration of 10 g/l after 24 hours caused the death of <50% of S. ransomi larvae. The study of flavouring additives with flowery odour, which are permitted to be used in food for humans and also to be used in cosmetics, is a promising field for research aimed at the development of new antiparasitic drugs.

Therefore determining the viability of helminths is significant for controlling their population, both in the host, and in the environment.Nowadays, synthetic antiparasitic drugs (Ponomar et al., 2013) are used and experiments of identifying the antihelminthic properties of plants are being conducted (Rahmann and Seip, 2006;Burke et al., 2009;Lu et al., 2010).
To fight pathogenic organisms, microbiologists and virusologists are researching the effect of flavouring agents in food production (Chiang et al., 2005;Sato et al., 2006;Somolinos et al., 2008;Si et al., 2009;Belletti et al., 2010).Because pigs consume food not only of vegetable origin, but also of animal origin, their diet can include synthetic flavouring agents from the diet of humans.Therefore the objective of this research is to define the effect of flavouring agents upon the level of viability of S. ransomi larvae (Rhabditida, Strongyloididae).

Materials and methods
The faeces of pigs were studied to find S. ransomi larvae using the Baermann test (Zajac et al., 2011).The material was collected in the summer period in Dnipropetrovsk district, and then transferred to the laboratory in plastic containers at a temperature of 22-24 °С. S. ransomi culture is represented by larval stages of freely moving and parasitic forms (Van Wyk and Mayhew, 2013).
Larvae of first and second stage (freely moving) have a rhabditelike oesophagus with two bulbuses.Their intestinal cells are not distinguishable.The parasitic form (third stage) is distinguished by a filariform oesophagus with no extensions; the intestine does not have cells which can be clearly distinguished through an optical microscope (Fig. 1).
After cultivation, the liquid with the larvae was put in test tubes (10 ml) by 4 ml and was centrifuged for four minutes at 1500 revolutions a minute. 1 ml of sediments with larvae was evenly weighed and put in 0.1 ml portions in plastic containers with a capacity of 1.5 ml.After that, 1 ml of the substance under research was added to the larval culture (20-40 ind.), which then was kept for 24 hours at a temperature of 22-24 °С.Four concentrations (10, 1, 0.1 и 0.01 g/l) of each of the flavouring agents were used in the experiments (Table 1), with eight replications.

Results
The flavouring agents citral, benzaldehyde exhibited the strongest effect on the viability of S. ramsomi larvae (Table 2).Note: «-» -the experiment did not achieve death of 50% larvae (needed concentration of more than 10 g/l).
D-limonene and beta-ionone at the studied concentrations (less than 10 g/m) did not affect the viability of S. ransomi larvae signifycantly, which shows that they are not useful as nematocidal medicines.The maximum effect upon the nematodes at LD 90 (Fig. 2а, c) was shown by benzaldehyde at 685 mg/l.In this case, the larvae of the 100% nematodes studied perished in benzaldehyde solutions with a concentration of 1 and 10 g/l.Thus, this substance can be used for further development of veterinary drugs with anthelmintic effect.

Discussion
Nowadays research is being conducted on the extent of effect of flavouring agents on the viability of agents of infection.Chiang et al. (2005) discovered the antiviral function of a broad spectrum of apigenin, linalool and ursolic acid.These substances are extracted from basil, which is familiar in Chinese medicine as a medicinal plant.The antimicrobial function of these compounds such as linalool and citral is well attested.They are capable of inhibiting the growth of pathogenic microorganisms (Sato et al., 2006;Somolinos et al., 2008;Si et al., 2009;Belletti et al., 2010).Research on the effect of citral on infectious agents also supports the findings from our experiments on the viability of S. ransomi larvae.The effect of benzaldehyde and beta-ionone has been insufficiently studied.
Methylparabene is used against fungi, and also as an antiseptic (Shapiro et al., 2002;Posey et al., 2005;Kromidas et al., 2006;Rebbeck et al., 2006;Ishiwatari et al., 2007;Gopalakrishnan et al., 2012).This substance is also included as a preservative in an insecticide aimed at controlling agricultural pests (Bell, 1990).The flavouring agent benzaldehyde also shows insecticidal properties.The negative effect of benzaldehyde upon Galleria mellonella (Linnaeus, 1758) has been demonstrated.Therefore it is recommended for use in developing new agricultural insecticidal preparations (Ullah et al., 2015).The effect of beta-ionone upon living organisms has received insufficient study.Indeed, the effect of entire groups of flavouring agents upon living organisms requires further studyin general.

Conclusions
We researched the effect of flavouring agents such as benzaldehyde, citral, D-limonene and beta-ionone upon the viability of S. ransomi, parasitic larvae on pigs.We determined the minimum values of LD 50 for benzaldehyde and citral.Experimentally, a death rate of 50% of the tested larvae was not achieved using D-limonene and beta-ionone.Food additives with a pleasant flower odour, which are permitted to be used for food, and which are used as cosmetics, are important for evaluating potential new antihelminthic medical and veterinary preparations.

Fig. 2 .
Fig. 2. The effect of benzaldehyde (а), D-limonene (b), citral (c) and beta-ionone (d) on the viability of S. ransomi larvae: along the axes of abscissa -variant of experiment (mg/l), along the axes of ordinate -viability of S. ransomi larvae during 24 hours of laboratory experiment (%); n = 8 for each variant of experiment

Table 1
Properties and usage of flavouring agents* which were used for establishing the viability level of S. ransomi larvae

Table 2
LD 50 (x ± SD) for S. ransomi larvae in laboratory experiment