Changes in the viability of the eggs of Ascaris suum under the influence of flavourings and source materials approved for use in and on foods

One of the most common parasitic diseases of pigs globally is ascariasis. It is caused by the nematode Ascaris suum Goeze, 1782 (Nematoda, Ascaridata), which parasitises the small intestine of pigs in its mature form and the respiratory system at the larval stage. This helminthiasis causes immense damage to swine-rearing. Control of the ascariasis pathogen in the host’s organism and in the environment is essential for the health of the animals and successful swinerearing. The results of studying the effect of flavourings and source materials approved for use in and on foods – cinnamaldehyde (0656 Codex Alimentarius), benzoic acid (Е210 Codex Alimentarius) and methylparaben (Е218 Codex Alimentarius) on the viability of invasive eggs of A. suum are useful for determining the minimum concentration of solution of these substances for use against eggs of A. suum (10 g/l). The lowest efficiency against invasive eggs of A. suum was obtained for methylparaben (LD50 = 3850 ± 2130 mg/l), the highest was obtained with cinnamaldehyde (LD50 = 2437 ± 864 mg/l), and benzoic acid (LD50 = 1240 ± 680 mg/l).


Introduction
Swine rearing is one of promising and developed spheres of livestock breeding in the world, and in Ukraine in particular (Feshenko, 2008;Evstafieva, 2010).There are a number of factors which negatively affect its development.First of all, these factors include parasitic diseases, such as ascariasis, trichocephalosis, oesophagostomiasis, strongyloidiasis, echinococcosis, cysticercosis, sarcoptosis, balantidiasis and others.The most widespread disease of pigs globally is ascariasis (De Velásquez et al., 2004;Feshenko, 2008;Evstafieva, 2010;Katakam et al., 2016).It causes great damage to swine-rearing.The prevalence of the disease on pig farms can reach 100%.The disease is especially harmful for young (pigs up to one year old).The intensity of their infestation can reach several hundred eggs in 1 g of feces.
Ascaris suum Goeze, 1782 affects not only the digestive system, but also the respiratory system.Infestation can cause intestinal disorders; high intensity of infestation among piglets causes intestinal blockage, rupture, therefore peritonitis and death.Hepatopulmonary migrations of A. suum lead to liver disorders and bronchopneumonia (Bila, 1999;Evstafieva, 2010).According to Ponomar and Soroka (2008), helminths produce special substances, which prevent the proliferation by T-and B-lymphocytes.Under the influence of helminths' antigens, a great number of T-suppressors appear in the host's organism, which decreases the activity of other subpopulations of T-lymphocytes.This leads to development of immunological tolerance among young pigs.Deficiencies in the immune system are a key factor which determines the onset and duration of the invasive process.Nematodes block the host's defense processes.One of the mechanisms of such immunosuppression is the phenomenon of antigen competition, when T-lymphocytes, which were activated by the helminths' antigens, suppress the ability of B-lymphocytes to produce antibodies to antigens of other infestation agents.This explains the complications during the infection process of A. suum infestation and the decrease in the level of postvaccinal immunity against infections, and often causes collapse in the immune system (Ponomar and Soroka, 2008).
Today, scientists all around the world are developing methods of utilization of swine manure, its disinfection, elimination of eggs of A. suum in the environment, and treatment of pigs against ascariasis.Recently veterinary specialists have tended to prefer complex injectional antihelmintic preparations, which are efficient both against endo and ectoparasites, and also against different stages of helminths' development.In this respect, preparations of macrocyclic lactone, which are efficient against nematode infestation, appear to be promising.Also there are antihelmintic preparations, which are efficient both at the larval and mature stages of parasites' development.Many Ukrainian and foreign authors (Beloeil et al., 2003;Veneziano et al., 2004;Fthenakis et al., 2005;Gudkova et al., 2006;Antipov, 2010;Artemenko, 2011) have reported the high antihelmintic efficiency of preparations of the benzimidazole group (albendazole, phenbendazolum).A number of researchers have conducted experiments on the influence of herbal preparations and flavourings upon the viability of nematode larvae (Chiang et al., 2005;Sato et al., 2006;Somolinos et al., 2008;Si et al., 2009;Belletti et al., 2010).

Materials and methods
In the experiment, we used fresh eggs of A. suum nematodes from swine feces, taken from farms in Dnipropetrovsk region.Samples of pigs' feces were delivered in plastic containers at the temperature of +17...+19 °С to the laboratory of the Parasitology and Veterinary-Sanitary Examination Department at the Dnipro State Agrarian-Economic University.At the beginning of experiment, A. suum eggs (Fig. 1) were detected by the McMaster method (Pereckiene et al., 2007;Zajac et al., 2011).A thick brown tuberous shell is typical for these eggs.Females of this nematode species lay grey eggs in the small intestine.During their way through the intestines, they obtain a brown colour due to the feces.The eggs detected were not mature: inside they had bleached blastomeres in the process of fragmentation, which is typical for eggs of this species of nematode.Average intensity of A. suum infestation in the selected material was 475 eggs/g of feces.Later in the study, swine feces were weighed by portions of 10 g.The samples were put in glasses, by addition of water, the eggs of A. suum were "washed" from the feces.The "washed" eggs were then put in a Petri dish and left for 21 days in a thermostat at the temperature of +28 °С for cultivation of larvae inside the eggs before the infestation stage (Fig. 2).Such eggs contained sedentary larvae, twisted in form larvae.At the same time, for preventing the breeding of bacteria and fungi in the sample, 2% solution of formalin was added.Then the solution with eggs was uniformly stirred and poured in 0.1 ml portions into plastic test-tubes of 1.5 ml capacity.Then 1 ml solution of flavourings was added: cinnamaldehyde (0656 Codex Alimentarius), benzoic acid (E 210 Codex Alimentarius) and methylparaben (Е 218 Codex Alimentarius).The experiment used three concentrations of the substances (10, 1 and 0.1 g/l), and also the control (distilled water); each experiment was conducted with eight replications (Fig. 3).The duration of the experiment was 24 hours at the temperature of +22...+24 °С.The viability of the larvae in the eggs was determined by their mobility when the temperature of the fluid containing the eggs was raised to +37 °С in the solutions of the studied substances.

Results
Laboratory experiments on the influence of flavourings and source materials approved for use in and on foods (cinnamaldehyde, benzoic acid, methylparaben) upon the viability of larvae and eggs of nematodes indicate that the concentration of these substances significantly contributed to the mortality of parasites.After raising the temperature of fluid containing the eggs to +37 °С in the solution of 10 g/l cinnamaldehyde concentration, no mobility of A. suum larvae in eggs was registered.Only 39% and 16% of A. suum larvae died under the concentrations of 1 and 0.1 g/l respectively.LD 50 for invasive eggs was 2 437 ± 864 mg/l (Fig. 3а).
Similar results were registered for pathogens of ascariasis after using benzoic acid.About 55% of larvae in eggs survived at 1 g/l concentration of this substance.With smaller concentrations of benzoic acid, we observed a 100% survival rate of larvae in the eggs of A. suum.Only at 10 g/l concentrations of benzoic acid did 100% of eggs die.For A. suum at the infestation stage LD 50 =1 240 ± 680 mg/l (Fig. 3b).Methylparaben was the least effective.Under 10 g/l concentration of methylparaben 20% of larvae in eggs of A. suum survived.At smaller concentration (1 g/l) the substance killed 25% of larvae in the eggs of nematodes.Methylparaben solution at 0.1 g/l concentration killed about 10% of larvae in eggs of A. suum.LD 50 for methylparaben at the infestation stage of A. suum eggs was 3 850 ± 2 130 mg/l (Fig. 3c).

Discussion
The search for the most effective methods of controlling ascariasis of swine continues to this day.For deactivation of the eggs of these nematodes, ammonia is used (Nordin et al., 2009;Katakam et al., 2014;Fidjeland et al., 2015).The temperature of NH 3 is an essential factor: increase in temperature speeds up the deactivation of A. suum eggs.Therefore Fidjeland et al. (2015)  recommend adding urea and other alkaline material when processing feces of pigs with ascariasis, and also advise raising the temperature.Similar experiments were also conducted by Katakam et al. (2014).According to the results of their research, following an increase in temperature, the time needed to deactivate A. suum eggs decreased.Nordin et al. (2009) proved the lethal action of urea and temperature +34 °C on eggs of A. suum.Analysis of their publications proves that the deactivation of nematode eggs in such conditions takes less than 10 days.For deactivating the eggs of A. suum in the environment, Nelson et al. (2001) recommend using a mixture of reagents: the observed deactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution.De Velásquez et al. (2004) have reported the lethal action of ozone on the eggs of A. suum.Under the influence of ozone, 90% of eggs became nonviable already after the first 60 minutes.Another 10% of the number of surviving eggs died after the second hour of the experiment.The concentration of dissolved ozone in the liquid phase (in the solution of feces) was from 3.5-4.7 mg/l.
In "organic" swine rearing, the usage of chemical substances against the pathogens of infections and infestations is strictly limited.Therefore pigs in such farms are more heavily affected by A. suum infestation."Organic" farms decrease the level of environmental contamination with A. suum eggs by regular mechanical cleaning of the pigs' living area from feces with further compulsory composting (Katakam et al., 2016).Katakam et al. (2014) in their experiments studied the relationship between viability of A. suum eggs in deep litter and the litter's humidity and temperature.At a temperature of +43.6 °C and relative humidity of 43% the viability of A. suum eggs was no higher than 5%, and only 0.004% of eggs developed to the larval stage.The influence of temperature on the development of A. suum eggs was described by Kim et al. (2012).A. suum eggs were unable to develop for a month at a temperature of 5 °C.The optimum temperature for the development of eggs of this nematode species is +35 °C: the larvae of A. suum develop in eggs already on the 17th day of incubation.
Decreasing the number of A. suum eggs in the environment is possible not only by processing the pigs' feces.This can also be achieved by increasing the resistance of the pigs' organism by using a balanced diet dominated by protein.Lack of iron and also the decrease of protein content in the diet leads to malnutrition, loss of weight, decreased resistance in the pigs' organism to A. suum (Pedersen et al., 2001(Pedersen et al., , 2002)).Williams et al. (2015) recommend using purified extract of Cichorium intybus Linnaeus, 1758 for phytotherapy of A. suum larvae.It can affect the viability of larvae of these nematodes in vitro.The extract inhibits glutathione-S-transferase activity of A. suum.
As an alternative to using synthetic antihelmintic preparations for decreasing the number of eggs which enter the environment through pigs' feces, and also for decreasing the level of pigs' infestation with A. suum, Kaplan et al. (2014) recommend using a mixture of essential oils: TTN1013 (a-pinene, linalyl acetate, p-cymene, and thymol octanoate) and TTN1014 (a-pinene, linalyl acetate, p-cymene and thymol acetate).
De Araújo et al. (2008) 2016) covers the study of the influence of volatile fatty acids (VFA) of four different compounds on the viability of eggs of A. suum.The compounds included acetic, propionic, butyric, valeric, and isovaleric acids.The studied concentrations of these acids are often found in acidogenic anaerobic digesters.According to the results of the experiment, the viability of A. suum eggs decreased after using these acids.

Conclusions
One of the most common parasitic diseases of pigs globally is ascariasis.Control of the ascariasis pathogen in the host's organism and in the environment is essential for the health of the animals and successful swine-rearing.According to the results of study on the effect of cinnamaldehyde, benzoic acid and methylparaben upon the viability of eggs of A. suum, we recommend using minimum concentration of solutions of these substances against the eggs of A. suum, at 10 g/l.
Fig. 2. Mature egg of A. suum on 22nd day of the experiment: pointer indicates the formed invasive larvae, bar = 10 µm

Table 1
Properties and usage of flavouring agents* which were used for establishing the viability level of A. suum larvae 3 White crystalline substance with a typical smell Is found in the roots of Oxalis tuberosa As a preservative As an antiseptic recommend using fungi-helmintophages Duddingtonia flagrans (Dudd.)R. C. Cooke, 1969, Monacrosporium sinense Xing Z. Liu & K.Q.Zhang, 1994, Pochonia chlamydosporia (Goddard, 1913) Zare et W. Gams, 2001 with domination of the latter species for biological control of the number of A. suum.Similar studies have been conducted by Ferreira et al. (2011): they also showed high ovicidal activity of P. chlamydosporia fungi against eggs of A. suum.The work of Rojas-Oropeza et al. (