Molecular evaluation of Toxocara species in stray cats using loop‐mediated isothermal amplification (lamp) technique as a rapid, sensitive and simple screening assay

Abstract Toxocara species are parasitic nematodes of dogs and cats with a worldwide distribution. The adult worm lives in the intestine, and horizontal transmission of the infection occurs through eating paratenic host or embryonated eggs. This study aimed to estimate the molecular prevalence of Toxocara species in stray cats using the loop‐mediated isothermal amplification (LAMP) technique. A total of 95 stool samples were randomly collected from stray cats in Khorramabad city in western Iran. Microscopic examination was performed after the separation and extraction of supernatants. The LAMP reaction was performed using the internal transcribed spacer 2 (ITS2) gene primers of Toxocara species and the appropriate master mix. The overall prevalence of Toxocara spp. in stray cats was 20% (19/95, CI 95%: 0.2 ± 0.08) by parasitological and molecular assessments. The microscopic examination of stool samples revealed that 19 samples were positive for Toxocara. The same 19 positive samples were also positive by the LAMP technique. Interestingly, based on the results of the LAMP assay, out of 95 studied samples, 18 (18.94%; CI 95%: 0.19 ± 0.08) specimens were Toxocara canis, while only 1 (1.05%; CI 95%: 0.005 ± 0.01) sample was diagnosed as Toxocara cati. The relatively high prevalence of Toxocara species in the studied cats shows the role of this species in spreading the parasite and the role of the cats in transmitting this zoonotic parasite. Preventive measures including the control of stray cat's population by castration and protection of public gardens where children play are recommended. The easy, highly sensitive and specific LAMP method is proposed for the differential detection of Toxocara species in animals and humans.


| INTRODUC TI ON
Ascarid nematods (Toxocara canis, Toxocara cati and Toxascaris leonina) are parasitic roundworms of predatory mammals from families Canidae and Felidae with worldwide distribution (Eslami, 2006;Okulewicz et al., 2012). Toxocara canis is one of the important public health and economically important zoonotic parasitic infections in humans sharing with dogs, somewhat cats and wild canids, particularly foxes and jackals (Lee et al., 1993). The definitive hosts of T. cati are wild and domestic felids (Okulewicz et al., 2012). The adult worm lives in the intestine of dogs and cats, and infection of humans is caused by the ingestion of embryonated eggs or raw or undercooked liver and/or meat from paratenic hosts including cattle, sheep, ducks, pigs, lambs and chicken (Acosta et al., 2011;David & Petrie, 2006;Maruyama et al., 1994). The highest prevalence of Toxocara is seen in kittens and puppies of 12-24 weeks of age (O'Lorcain, 1994). Puppies may be infected by breast milk or through the umbilical cord during fetal development, kittens are also infected through breast milk (Athari, 2010;Nabavi et al., 2014;Rubinsky-Elefant et al., 2010). The zoonotic disease resulted from these nematodes is called toxocariasis (Durant et al., 2012). Toxocara species do not mature in the human body, however, contamination with the second stage larvae (L2) can lead to serious complications such as high eosinophilic visceral larvae migrans (VLM), hepatomegaly, chronic pulmonary inflammation associated with cough and fever which can sometimes lead to breathing problems and even death (Glickman & Schantz, 1981;Kumagai et al., 2010). Toxocariasis may also cause ocular larvae migrans (OLM) with eye disorders as well as epilepsy and myocarditis (Alavi & Sefidgaran, 2008;Glickman & Schantz, 1981;Kumagai et al., 2010).
Toxocara human infection has been reported in almost all regions of the world, especially those with tropical weather and abundant dog and cat populations. Approximately, 10% of the human world population are seropositive for toxocariasis (Lappin, 1993). In addition to developing countries, in some developed countries such as Germany, Hong Kong, China and Australia, toxocariasis is one of the most common human helminthic infections. Human infections are more commonly reported in children, especially children of low age (Despommier, 2003;Glickman & Schantz, 1981). Keeping dogs (especially puppies) and cats as a pet and touching and playing with these animals, geophagy by children, free entry of dogs and cats into farmland and public parks, and non-compliance with sanitation in eating non-washed vegetables are among the most important risk factors associated with toxocariasis (Rubinsky-Elefant et al., 2010).
Toxocara infection can be detected in dogs and cats using epidemiological and clinical elements and coproscopy. The wet mount (direct smear) preparation from the stool specimen solely has low sensitivity for finding eggs while accompanied by the concentration methods such as flotation assay make a more accurate diagnosis (Dryden, 1996). The sensitivity and specificity of the floatation method for detecting Toxocara eggs were estimated to 51% and 100%, respectively (Overgaauw, 1997). The use of new diagnostic methods, such as molecular assays, to estimate the prevalence of infection in dogs and cats as reservoirs of Toxocara, is applicable and can be compared to the results of other tests, including routine stool examination. The loop-mediated isothermal amplification (LAMP) technique is a simple high-performance method that was developed by Notomi et al. (2000), in which the DNA replicated specifically, efficiently, and rapidly under isothermal conditions (Fallahi et al., 2014;Notomi et al., 2000). In this method, four specific designed primers (two internal and two external primers) that identify six specific regions among the target DNA are used Mirahmadi et al., 2020). The target region is replicated during the comet process, forming a series of the loop at a temperature of 60°C-65°C by using a thermoresistant DNA polymerase enzyme (Bst DNA polymerase). The LAMP technique is a simple method that does not require expensive equipment such as thermal cycler and gel documentation systems and the reaction can be done in a hot water bath or thermal block (Fallahi et al., 2015(Fallahi et al., , 2018Ghodrati et al., 2017;Ghodsian et al., 2019;Hanifehpour et al., 2019;Valian et al., 2020). The results can be simply evaluated by visual examination of the tubes and the colour change of SybrGreen I, an intercalating dye that specifically binds to the double-stranded DNA in reaction tubes from orange to green fluorescence under daylight and UV light (Arab-Mazar et al., 2019;Fallahi et al., 2020;Kaneko et al., 2007;Mori et al., 2007;Nagamine et al., 2001).
The high population of stray cats, as one of the definitive hosts of Toxocara, in Khorramabad city, Western Iran, the low number of studies conducted in this area, and the high resistance of Toxocara eggs, as a major source of environmental contamination, emphasizes the need for research on the prevalence of Toxocara in stray cats.

| Samples collection
During the 3-month period (July-September 2018), a total of 95 stool samples were randomly collected from stray cats in Khorramabad city, Lorestan province in west of Iran. Stool samples were immediately transferred to the Parasitology Laboratory Faculty of Medicine, Lorestan University of Medical Sciences, Iran, and divided into two parts. One part of each specimen was prepared for parasitological examinations, and the other part was kept at −20°C for DNA extraction and subsequent molecular evaluation.

| Parasitological examinations
The stool specimens were poured into a tube containing isotonic saline (0.85% NaCl solution) and vortexed for 5 min. The homogenized samples were examined by microscopic observation (Zeiss, Germany, ×100 and ×400 magnification) of direct smears using isotonic saline, and floatation with a saturated chlorine solution. To separate the debris, the suspensions were passed through two layers of wet tampons. Thereafter, the samples were centrifuged at 3,000g for 3 min, the supernatant was discarded and the saturated chlorine solution was spilled into the tube and centrifuged at 2,000g for 5 min. Then, 1.5-ml microtube. Toxocara eggs were identified by microscopic observation of supernatant under ×100 and ×400 magnification.
To rupture Toxocara eggshell and allow DNA extraction, sonication and freezing-thawing methods were used. First, the sonication cycle of the sonicator machine was set to 0.5 cycles, and the voltage to 70 V. The microtubes containing the specimens were placed inside the ice and subsequently, each sample was sonicated for 20 s (5 times every 4 s). Subsequently, the samples were placed five times every 2 min inside the liquid nitrogen then water bath at 95°C.

| Parasitological examinations
Based on parasitological examinations of the stool specimens by direct smears (wet mount) and floatation with a chlorine solution, the prevalence of Toxocara spp. was 20% (19/95; CI 95%; 0.2 ± 0.08) among stray cats of Khorramabad in western Iran (Figure 1). The number of positive samples detected by the flotation and wet mount direct smears methods were 12 and 7, respectively (data not shown).

| D ISCUSS I ON
Considering the effect of Toxocara spp. on human health including VLM and OLM syndromes, hepatomegaly, pulmonary involvement, myocarditis, epilepsy and even death, as well as the high pathogenicity in cats and dogs, specially puppies, detection of parasites in these animals, as definitive hosts of the parasites is of great importance (Kleine et al., 2017;Kumagai et al., 2010). Based on the results of the present study, 20% (19/95; CI 95%: 0.2 ± 0.08) of the studied cats were infected by Toxocara spp., among them, 18 animals (18.94%; CI 95%: 0.19 ± 0.08), were infected by T. canis while only one animal (1.05%; CI 95%: 0.005 ± 0.01) was infected by T. cati. These results indicate the relatively high prevalence of Toxocara species, especially T. canis, in stray cats in Khorramabad in western Iran, which shows the potential role of these animals as important reservoirs for other animals and humans, especially children. The results of the present study are consistent with those carried out in different regions of Iran on cats in terms of a relatively high Toxocara prevalence (Arbabi & Hooshyar, 2009;Changizi et al., 2007;Khademvatan et al., 2013;Sharif et al., 2007;Torkan et al., 2017). However, contrary to other studies, the dominant species in the current study was T. canis.
Such result could be due to the high prevalence of stray dogs in Khorramabad city in western Iran, high contamination of the environment, and possibly the transmission of infection to stray cats. There have been two reports from Malaysia of a 'variant' ascaridoid of cats assigned to T. canis (Lee et al., 1993;Rohde, 1962). Lee et al. (1993) reported that of the 55 examined cats from Kuala Lumpur suburbs, Malaysia, 15 were infected by Toxocara spp. of these, 12 were infected by T. cati and the others by T. canis. Likewise, Parsons and colleagues (1988) (2016) showed a higher sensitivity of LAMP (detection limit 1-3 eggs/200 g soil) than PCR (detection limit > 3 eggs/200 g soil) and microscopy techniques.
The regional difference of Toxocara prevalence could be ex-

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest. Writing-original draft.

E TH I C A L S TATEM ENTS
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The study was approved by the ethical committee of the Lorestan University, Khorramabad, Lorestan Province, West of Iran.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.431.