Sodium hypochlorite as an Artificial Exsheathment Medium for Nematode Larvae: Limitations for Application

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Sodium hypochlorite as an Artificial Exsheathment Medium for Nematode Larvae: Limitations for Application

https://doi.org/10.21203/rs.3.rs-2748755/v1

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Sodium hypochlorite (NaOCl) has largely been used as an artificial exsheathment medium for nematode larvae in various studies such as evaluating the efficacy of anthelmintic drugs, assessing the resistance of nematodes to anthelmintics, evaluating plant bioactive products as anthelmintics, or in nematode viability assays because of its unique capacity for tissue dissolution. Doubts, however, exist that the compound affects the viability of the exsheathed larvae. The present study’s objective was to exclude or confirm possible negative effects of the compound using strongylid third-stage larvae of donkeys’ origin. Visual larval immobility and gross morphological changes were adopted as criteria for assessment of viability following treatment of larvae with 0.025, 0.05, 0.1, 0.2, 0.3, or 3% NaOCl solutions. Larvae were examined 10, 5, or 3 min after treatment. The results indicate there is a threshold beyond which viability is impaired. A concentration of 0.025% produced no effect on the viability of larvae (the optimum concentration) for as long as 10 min following treatment (the effective time). It is proposed that in situations where the optimum concentration has been determined, it is necessary to establish if exsheathment can be produced within the respective effective time limit.

sodium hypochlorite

exsheathment

strongylid larvae

tissue dissolution

limitations

viability

Sodium hypochlorite (NaOCl) is an ionic chemical compound with a unique capacity for tissue dissolution (Echeverri and Acuña 2012; de Almeida et al. 2013; Khoshroo et al. 2020). The compound has largely been used as an artificial exsheathment medium for nematode larvae in various studies such as evaluating the efficacy of anthelmintic drugs (Campbell and Gaugler 1991; Patel and Campbell 1997; Feyera et al. 2022), assessing the resistance of nematodes to anthelmintics (Almeida et al. 2013; Bihaqi et al. 2020; Potârniche et al. 2021), evaluating plant bioactive products as anthelmintics (Arau´jo et al. 2017; Jamous et al. 2017; Reséndiz-González et al. 2022 ), or in nematode viability studies (Gómez-González et al. 2021). Coles et al. (1980) compared different media (viz acid pepsin, ox bile, sodium tetraborate, and NaOCl) as exsheathing agents for nine species of nematodes. They described NaOCl as the only satisfactory exsheathing medium. Conder and Johnson (1996), on the other hand, used the Mongolian jird (Meriones unguiculatus) as an experimental model to evaluate the viability of exsheathed larvae of ruminant parasites following exposure to different media (distilled water, Earle’s balanced salt solution + CO₂, nematode washing buffer + CO₂, NaOCl) taking infectivity as a measure of viability. They found that NaOCl, although capable of providing a high percentage of exsheathment (≥ 98.5%), the infectivity of the exsheathed larvae produced was significantly lower than that of larvae exsheathed in the other media. They concluded that when exsheathing agents are used, the results should be interpreted with caution since the techniques commonly recognized to be highly effective “also appear to reduce viability”. In a previous study (Elowni et al. 2022), we used Strongyloides papillosus, a nematode whose larvae naturally lack a protective sheath, as a model to exclude or confirm possible negative effects of NaOCl on the viability of larvae. The results indicated that the effect of the compound is dependent upon both the concentration (C) and the time of exposure (T) to the compound and there are certain limits (0.3% ˃ C ˃ 0.2%; 10 min ˃ T ˃ 5 min) beyond which viability is impaired. Generally, NaOCl had been used as an exsheathing agent at different concentrations ranging from 0.005% in some studies to an absolute 3.5% solution in others. Likewise, the compound’s application period also varied considerably between investigations ranging from < 1.0 min up to 18 hrs. The present study’s objective was to determine the optimum concentration of the compound that can be used without compromising larval viability taking the Strongyloides model results as guidelines. Third-stage strongylid nematode larvae of donkeys’ origin were used for this purpose. Generally, nematodes' viability is assessed by observing different parasite phenotypic characteristics including gross morphology, developmental processes, or motility (O’Grady and Kotze 2004; Smout et al. 2010; Andre et al. 2016; Preston et al. 2016) with motility being the most commonly adopted criterion (Bennett and Pax 1986; Smout et al. 2010), commonly by visual means (Mackenzie et al. 2017). In this study, visual larval motility and gross morphological changes were used as a measure for the assessment of the effect of NaOCl on viability.

Source of parasites and culture of larvae

Fresh feces were collected from naturally infected working donkeys and were transported in screw-cap bottles to the Parasitology laboratory at the Faculty of Veterinary Medicine, University of Khartoum. Samples were screened for strongylid eggs by flotation technique using sodium chloride hypertonic solution. Fecal masses containing eggs were thoroughly crumbled, moistened sufficiently with tap water, and cultured at room temperature (30 - 33 °C) for 9 -10 days to allow complete larval development to the infective third larval stage. The larvae were retrieved from culture by the Baermann standard technique. Random larval samples were examined microscopically using the number, arrangement, and shape of intestinal cells (Santos et al. 2018; Maestrini 2020; Amer et al. 2022) as criteria for generic/species culture composition.

Experimental procedures

In all trials, the larvae harvested in eluent culture fluid were transferred to centrifuge tubes and centrifuged at 2,000 rpm for 2 minutes. The supernatant was decanted leaving a trickle of fluid at the bottom of the tubes. A drop of suspended larvae is deposited on a glass microscope slide to which 10 μL of NaOCl (AMCLEAN, Al Fayrouz Dental and Medical Equipment, Sharjah, UAE) of different concentrations was added using a micro-volume pipetter (Accumax, India). The mixture was covered with a glass coverslip and the larvae were examined at ×40 and ×100 magnifications using an OPTIKA Srl B-193 compound microscope (Ponteranica, Italy) fitted with a digital camera (OPTIKAM 4083.B1).

Effect on motility and gross morphology of larvae

The viability of larvae was assessed by evaluating the status of individual larvae, regardless of the species present, 10, 5, or 3 min following treatment with 0.025, 0.05, 0.1, 0.2, 0.3, or 3% NaOCl solution. Trials were replicated using larvae from different cultures. Larvae were classified as actively motile, as compared with untreated controls (Supplementary movie file 1 (mp4); retrievable online from figshare 10.6084/m9.figshare.22324450), sluggish regardless of the degree, immotile, or damaged.

Larvae recovered from culture

The larvae retrieved from culture were of mixed strongylid species (Figs 1A - C) as indicated by the presence of larvae with well-defined triangular intestinal cells (Fig 1D), rectangular cells (Fig 1E), elongated cells (Fig 1F), irregular cells (Fig 1G), poorly defined cells (Fig 1H), or trapezoidal cells (Fig 1I). The cultures also contained Strongyloides westeri larvae verified by the distinctive long filariform oesophagus of almost 1/2 the total length of the larva, and the absence of a sheath (Fig 2).

Pilot tests

Larvae were treated on microscope slides with 10 μL of a 3% NaOCl solution as a benchmark to assess the initial dissolution capacity of the compound in terms of the compound’s exsheathing ability, effect on the motility of larvae, and effect on larval tissue. Exsheathment was a rapid process that occurred within 1 min following the application of the compound (Fig 3A). The dissolution capacity was manifested by a progressive dissolving effect on the cast sheath (Figs 3A-C). The chemical was potentially capable of reducing motility and inducing structural damage when the larvae were examined 10 min after treatment (Fig 4).

Assessment of viability

There was a reduction in motility when the larvae were exposed to chemical concentrations ˃ 0.025% for 10 min (Table 1). At the extreme 3% concentration, the majority of larvae (84.2%) were either sluggish or immotile, 10.5% were actively motile and 5.3% were structurally damaged. The response of larvae exposed for either 5 or 3 min almost followed a similar pattern (Tables 2 and 3, respectively) except that no gross larval damage has been observed in the group treated with a 3% solution. At the 0.05 % concentration, the overall performance of larvae tested after 10, 5, or 3 min indicated that 6.02% of the 415 larvae examined were sluggish while the rest were actively motile.

Table 1 Viability of 3^rd stage nematode larvae 10 min after treatment with NaOCl

	NaOCl concentration
	0.025%	0. 05%	0.1%	0.2%	0.3%	3.0%
=	(+)	(+, ± )	(+, ±)	(+, ±)	(+, ±, -)	(+, ±, -) •
r	4	4	4	5	7	4
n	54	59	32	56	134	57

(+) Actively motile, (±) Sluggish, (-) Immotile, (•) Larval damage

(=) Overall assessment (r) Replicates (n) Total number of larvae examined

Table 2 Viability of 3^rd stage nematode larvae 5 min after treatment with NaOCl

	NaOCl concentration
	0.025%	0. 05%	0.1%	0.2%	0.3%	3.0%
=	(+)	(+)	(+, ±)	(+, ±)	(+, ±, -)	(+, ±, -)
r	4	4	4	4	5	4
n	53	61	60	94	123	54

(+) Actively motile, (±) Sluggish, (-) Immotile

(=) Overall assessment, (r) Replicates, (n) Total number of larvae examined

Table 3 Viability of 3^rd stage nematode larvae 3 min after treatment with NaOCl

	NaOCl concentration
	0.025%	0. 05%	0.1%	0.2%	0.3%	3.0%
=	(+)	(+, ±)	(+, ±)	(+, ±)	(+, ±, -)	(±,-)
r	3	5	3	3	3	3
n	142	295	79	100	78	58

(+) Actively motile, (±) Sluggish, (-) Immotile

(=) Overall assessment, (r) Replicates, (n) Total number of larvae examine

NaOCl received wide application as an artificial exsheathing agent for larvae in nematode-based research (Bihaqi et al. 2020; Gómez-González et al. 2021; Potârniche et al. 2021; Feyera et al. 2022; Reséndiz-González et al. 2022) due to its remarkable tissue dissolution capacity, a property that has been proven experimentally when the compound was tested against a wide range of biological tissues such as bovine tendon collagen (Nakamura et al. 1985), bovine muscle tissue (Dutta and Saunders 2012), porcine muscle tissue (Echeverri and Acuña 2012), the bovine dental pulp (de Almeida et al. 2013), and its efficacy as an irrigant in endodontic medical therapy (Khoshroo et al. 2020). Nematodes in general form a diverse phylum including parasitic species which are of great medical, veterinary, and agricultural importance. The body is encased in an exoskeleton (known as the cuticle), a complex protective extracellular matrix covering the outermost layer of cells composed primarily of proteins with trace amounts of carbohydrates and lipids (Fetterer and Rhoads 1993; Page et al. 2014). NaOCl has commonly been used as exsheathing agent for nematode larvae at different concentrations ranging from 0.005% in some studies up to 3.5% in others. Likewise, the time of exposure to the compound also varied considerably between investigations ranging from < 1.0 min to 1 or 18 hrs. The exsheathed larvae are repeatedly washed to remove the chemical residues before they are used for experimentation. Therefore, it would be expected that a compound as NaOCl with such an effective tissue-dissolution capacity would have an adverse effect on the exsheathed larvae had they been overexposed to the compound. The present study’s objective was to determine the optimum concentration that can be used without compromising the viability of larvae.

The nematodes that normally parasitize equids fall into 7 suborders, 12 families, 29 genera, and 83 species with the great majority being members of a single family, the Strongylidae (Lichtenfels et al. 2008). The problem of isolating a pure mono-specific larval sample from a heterogeneous mixed culture (this study) makes it difficult to draw unreserved conclusions on the effect of treatment on viability. Regarding this difficulty, we resorted to assessing the response of individual larvae, regardless of the species, in replicated trials with larvae taken from different cultures. The results obtained are consistent with earlier findings (Elowni et al. 2022), using Strongyloides papillosus 3^rd stage larvae, that the effect of NaOCl is dependent upon both the concentration and the time of exposure to the compound. In this respect, Garduńo et al. (2010), using NaOCl concentrations of 0.003% to 1.3%, examined the effect of the compound on infective larvae of the sheep nematodes Haemonchus contortus and Cooperia curticei. They found that exsheathment occurred when the compound was applied at concentrations > 0.05% (0.26, 0.53, and 1.3%). However, at concentrations of 1.3% and 0.53 %, the larvae were killed within 1 and 2-3 hours, respectively. Similarly, Campbell and Gauler (1992) studied the effect of NaOCl on the entomopathogenic nematode Heterorhabditis bacteriophora. They found that nematodes exsheathed with a 1% solution for 5 minutes were significantly less motile than the normal sheathed larvae. They proposed that this decrease in motility may be the result of an ‘adverse effect’ of the compound on the larvae. Both findings by Campbell and Gauler (1992) and Garduńo et al. (2010) conform to the results of the present study that there is a threshold beyond which the viability of NaOCl-exsheathed larvae is reduced. The present results also indicate that, besides the effect on motility, the larvae treated with a 3% NaOCl solution become liable to damage in 10 min if not earlier. This probably explains why infective larvae of nematodes (H. contortus, Ostertagia ostertagi, Trichostrongylus colubriformis) exsheathed by exposure to NaOCl for 1 or 18 hours (Conder and Johnson 1996) have a low rate of infectivity when inoculated into Mongolian jirds.

The results described in this work are intended to serve as guidelines: the effect of NaOCl is dependent upon both the concentration and the time of exposure to the compound. There is a threshold beyond which the viability of larvae is impaired. A concentration of 0.025% produced no effect on the viability of larvae (the optimum concentration) for as long as 10 min following treatment (the effective time). It is proposed that in situations where the optimum concentration has been determined, it is necessary to establish if exsheathment can be produced within the respective effective time limit.

Funding

The authors did not receive support from any organization for conducting this study.

Competing interests

The authors have no competing interests to declare that are relevant to the content of this article.

Availability of data and material

All the data generated in this research are included in the main text of the article. Additional details, if any, will be provided upon request.

Authors' contributions

Elsayed E. Elowni conceived, designed, and performed the experiments, wrote the original manuscript, photomicrography. Ghada H. Abdelnabi and Mohamed F. Ahmed contributed equally to this work. Both prepared study materials, performed the experiments, interpretation of results. All authors read and approved the final manuscript.

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Sodium hypochlorite as an Artificial Exsheathment Medium for Nematode Larvae: Limitations for Application

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