Toxocara cati and T. canis are parasitic nematodes of feline and canine hosts respectively, that have a direct life cycle, and may include paratenic hosts such as rodents, rabbits, and birds, with occasional aberrant migration occurring in humans (Lee et al., 2010). The life cycle involves, unembryonated eggs exiting the infected dog or cat via fecal material, where the eggs, under the right conditions, including proper oxygenation, moisture and temperature, develop to eggs containing a third-stage infectious larva (L3). Once the next host, definitive or paratenic, ingests the infectious egg, the L3 hatch from the egg within the intestine, then migrate through the intestinal wall and into the circulation that carries them to the liver, then the lungs, where they are coughed up and swallowed. The L3 either grow to an adult worm in the small intestine of the definitive host or remain as a third-stage larvae in the tissues of the definitive, paratenic host, or accidental human host (Lee et al., 2010, Strube et al., 2013, Ahm et al., 2014). Canid or felid definitive hosts are infected either through ingestion of larvated eggs in soil, or through predation of infected animals containing encysted L3. Humans are infected following ingestion of the embryonated egg found on contaminated food, surfaces, soil, or ingestion of L3 within undercooked meat. While many people infected with Toxocara spp. experience no clinical signs, in severe cases, the larvae can travel to the major organs, to the eyes, or to the central nervous system causing toxocarisiais (Lee et al., 2010, Chen et al, 2018, CDC, 2019). Globally, it is estimated that more than 100 million dogs and about 118–150 million cats are infected with Toxocara spp. (Rostami et al., 2020a, Rostami et al., 2020b). Within the intestines of these hosts, female worms are prolific, producing approximately 200,000 eggs daily, approximately 27 million eggs in a 4-month lifespan, which then accumulate extensively in the environment via the feces, particularly in soil and sand (Shalaby et al., 2011, Miro and Bowman, 2018, Maciag et al., 2022). While most eggs will die within 6 months, under favorable environmental conditions, infectious Toxocara spp. eggs can remain in the soil for several years, and through environmental dissemination from actions such as foot traffic, rain, wind, and ingestion by earthworms, result in further soil contamination and increased potential for transmission including human infection (Mizgajska, 2001). The prevalence of Toxocara spp. eggs and the ability of Toxocara spp. eggs to endure for long periods of time within an environment necessitates the development of effective inactivation methods against these eggs to limit the prevalence of toxocarosis amongst humans and other animals.

T. vitulorum, a parasitic nematode of cattle, buffalo, and zebu, has a cosmopolitan distribution and is of economic importance (Ziegler and Macpherson, 2019). Although the life cycle is direct, transmammary is the main transmission route for calves while adults are infected by ingesting the embryonated eggs. Like T. canis and T. cati, upon ingestion of the embryonated egg, the larva hatches and performs a liver-lung migration. As adults, T. vitulorum worms reside in the small intestine and are found mainly in suckling and fattening calves. Depending on the worm burden clinical presentation manifests as diarrhea, emaciation, obstruction, and even death. Acquired immunity by older calves and adult animals results in resistance to patent infections, yet they may harbor third-stage larvae arrested within their tissues. The arrested larvae are reactivated in female cattle during pregnancy and migrate to the mammary glands and are passed to suckling calves where the larvae travel to the small intestine, mate and shed eggs via the feces into the environment, completing the life cycle (Anwar and Chaudry, 1984, Woodbury et al., 2012, Craig, 2018, Ziegler and Macpherson, 2019). Similarly to T. canis and T. cati, T. vitilorum may migrate in the tissues of aberrant hosts including rabbits guinea-pigs, rats, mice and chickens (Atallah et al., 1974) as well as the brains of mice (van Gorp et al., 1987). Conversely, T. vitulorum is less likely to persist in the tissues beyond 6 months (van Gorp et al., 1987). Yet, T. vitulorum larvae were detected in the milk of 13 out of 50 naturally infected bovines (26%), making this a source of potential risk to people that drink unpasteurized or unheated milk (Dewair and Bessat, 2020). A potentially confounding factor with serum surveys is that specificity needs to be validated as cross reactivity was demonstrated when T. vitulorum antibodies in serum of infected cattle was detected when using T. canis TES (van Gorp et al.,1987). This cross reactivity along with T. vitulorum tissue migration behavior gives rise to question whether T. vitulorum can cause human toxocariasis, however, this roll has not been established (Ziegler and Macpherson , 2019). T. vitulorum transmission may be of less concern regarding the human population altogether; however, for people in close contact with cattle or drink unpasteurized milk there may be an increased risk. Nevertheless, mitigating T. vitulorum transmission in animals is a public health concern and of economic interest.

Toxocara spp. eggs are incredibly resistant to both chemical and environmental factors due to their multi-layer shell composition, including a thin uterine membrane of acid mucopolysaccharide protein deposited by the female adult worm post fertilization, the thin electron dense pellicula ovi, a thick and homogeneous chitinous layer consisting of a chitinous microfibril core providing rigidity and physical protection, an electron-dense granular layer, and a flexible lipid dense permeability barrier membrane providing protection from desiccation and osmotic changes (Wharton, 1980, Bird and Bird, 1991, Ayçiçek et al., 2001, Bond and Huffman, 2023). Previous work with other nematodes has shown that there could be significant differences in resistance between the unembryonated egg and embryonated egg from changes in the egg’s membrane prior to hatching because of the enzymatic activity that occurs during larval development (Maya et al., 2012). An effective inactivation method against Toxocara spp. eggs must overcome the eggshell structure for both unembryonated and embryonated eggs; some methods tested include chemical, temperature, physical, and humidity changes.

Crucially, moisture is required for Toxocara spp. egg development and survivability. Although Toxocara spp. eggs can survive in soil with as little as 4.1% moisture content the optimum is 51-59% moisture content (Onorato 1932, Gamboa, 2005, Maya et al., 2010, Erofeeva and Vasenev, 2020). Unembryonated T. canis eggs did not embryonate at a relative humidity of 38% and temperature of 24oC, yet when the relative humidity was increased to 65% the eggs did embryonate (Onorato, 1932). Starting with either unembryonated or embryonated eggs, inactivation increases when humidity decreases; however, unembryonated eggs were less affected than embryonated eggs (Maya et al., 2010, Maya et al., 2012). Temperature also affects the vitelline membrane in helminth eggs and therefore affects survivability as well as embryonation (Maya et al., 2010). The optimum temperature for the eggs of T. cati and T. canis embryonation is between 15-30oC, however starting at a temperature of 34oC, a decrease in viability was observed, while a temperature of 37oC proved lethal, even though development had started (Onorato,1932, Gamboa, 2005, Maya et al., 2010, Abou-El-Naga, 2018). Temperatures below (-)32oC were also lethal (Owen, 1930). T. vitulorum eggs optimal embryonation temperature range is 20- 30oC, while temperatures from 1- 10C and above 36oC no development occurs. A temperature of (-)20oC for 2 hours is lethal to T. vitulorum eggs (Enyenihi, 1972, Usharani Devi et al., 2001, Anwar and Chaudhry,1984, Roberts, 1989). Another crucial requirement for embryonation is an exogenous supply of oxygen (Wharton, 1979). While development outside optimal conditions of moisture, temperature and oxygen may occur, the development is slowed, and the percentage of eggs reaching the infectious stage is negatively impacted (Wharton, 1979, Mizgajska, 2001, Erofeeva and Vasenev, 2020).Literature evaluating inactivation efficacy utilizing chemicals with T. cati or T. canis have included chemicals such as sodium hypochlorite (household bleach), glutaraldehyde, benzalkonium chloride, potassium permanganate, ethyl alcohol, potassium hydroxide, phenol, formalin, iodine solution, quaternary ammonium compounds, anionic and ionic surfactants, hydrogen peroxide and combination mixtures (Jaskoski, 1954, Ayçiçek et al., 2001, Morrondo et al., 2006, Zibaei et al, 2007, Shalaby et al., 2011, Verocai, 2010, von Dohlen et al., 2017, Romero et al., 2020, Ursache et al., 2020). Many of the chemicals tested with T. canis or T. cati, only evaluated inactivation with one status of egg, either unembryonated or embryonated, with most treatments being less than 100% effective. Most of the effective chemical treatments, such as surfactants (Jaskoski, 1954) and oxidizers (Shalabey etl., 2011) require at minimum one day of continuous exposure, except 7.5% iodine solution which was 100% effective against embryonated eggs after only 40 minutes continuous exposure (Ayçiçek et al., 2001). Studies with T. vitulorom have evaluated Lysol® a quaternary ammonium compound, tyrosinase and protease enzymes, flubenazole an anthelmintic, and formalin. Of which only 3% Lysol® was effective at 15 and 60-minute exposures with embryonate and unembryonated eggs respectively (Chauhan, et al 1980, , Singh, 2007, Shalaby et al., 2020). Previous work specifically with sodium hypochlorite has demonstrated that 0.1% sodium hypochlorite with a one-hour exposure at room temperature was ineffective in preventing embryonation to infectious stage eggs and even after 2 hours in undiluted house bleach 5.25% sodium hypochlorite T. canis eggs were able to embryonate and the larvae were motile indicating larvae were viable and potentially infectious (Ursache et al., 2020, von Dohlen et al., 2017).

Although there are some reported effective inactivation treatments for Toxocara spp., the contact treatment time is impractical for most surfaces being treated, and for routine use (Jaskoski, 1954, Ayçiçek et al., 2001, Morrondo et al., 2006). Despite sodium hypochlorite’s relative ineffectiveness to inactivate Toxocara spp., the wide availability of sodium hypochlorite in many household cleaners makes it a readily available resource. Therefore, using sodium hypochlorite treatment in combination with another reportedly unsuccessful treatment may work synergistically, resulting in an effective inactivation method for Toxocara spp. eggs. This study aims to determine whether the use of sodium hypochlorite in combination with desiccation is an effective inactivation method against Toxocara spp.