An alternative method for producing Toxocara canis second stage larvae from a paratenic host (pigeon) for mRNA extraction purpose

Abstract

Toxocara canis is a prevalent zoonotic parasite which can cause serious disease in puppies and humans. Excretory–secretory and coating antigens of the second stage larvae (L2) are the best targets for performing immunodiagnostic and also immunoprophylactic tests. Various hatching methods have been described to bring out L2 from the resistant infective egg shell; but these methods are difficult to do and have had different results when performed by different practitioners. In this study, second stage larvae were obtained from the viscera of pigeons (a paratenic host) which were infected with infective eggs. Infective Toxocara canis eggs were given to ten pigeons and live larvae were recovered from their excised livers and lungs by using the Baermann’s apparatus in the next days. Two in vitro methods for larvae hatching were also performed including a so-called physiological hatching method according to Ponce-Macotela et al. (J Parasitol 175:382–385, 2010), and a mechanical hatching method according to Alcântara-Neves and Santos (J Exp Parasitol 119:349–351, 2008) and their results were compared with the in-vivo method. Results show that averagely 36.2 % of fed larvae recovered from livers and 0.15 % from lungs. Average larvae recovery in the first day after infection (24.2 %) was significantly lower than subsequent days (39 %). Maximum larvae recovered in day 3 (55 %). In-vitro methods we carried out did not have acceptable results and only a few larvae did hatch using these methods.

Introduction

Toxocara canis is a widespread gastrointestinal nematode parasite of dog and some other canines which can cause serious disease in puppies. It is also a causative agent of Visceral and Occular Larva Migrans syndromes in humans (Lewis and Maizels 1993). Regarding the high serologic prevalence of Toxocara canis infection in human, and the fact that the disease is asymptomatic in most cases (Kayes 1997), numerous surveys have been performed to find proper immunodiagnostic and also immunoprophylactic methods in human and animals. Somatic antigens of the adult worms (Concepcion and Barriga 1985; Abo-Shehada et al. 1991), and the second stage larvae (L2) (Barriga 1988), the excretory–secretary antigens of larvae (TES) (Dvorožnáková et al. 2002; Sugane et al. 1996; Page et al. 1991) and also the recombinant antigens of larvae (Loukas et al. 1999; Maizels et al. 2000; Mun-Yik and Yee-Ling 2004) have been used for the above purposes. Limitations due to presence of cross-reactive antigens in TES and also in regularly producing sufficient quantities of a standardized TES product, have led researchers to investigate the possibility of using recombinant TES antigens for serodiagnosis (Holland 2006). The recombinant antigens TES-30 (Yamasaki et al. 1998) and TES-120 (Fong et al. 2003) are used for immunodiagnosis in human and TES-70 (Loukas et al. 2000) and Tc-Muc-5 (Doedens et al. 2001) are used in mice.

The arrested L2 have been shown in mammals such as mouse (Epe et al. 1994; Jin et al. 2008; Ishiyama et al. 2009; Dubey 1968), gerbil (Alba-Hurtado et al. 2009), cattle (Fitzgerald and Mansfield 1970; Yoshikawa et al. 2008) and pig (Helwigh et al. 1999); and also in birds such as poultry (Galvin 1964; Beaver 1956) and quail (Pahari and Sasmal 1991). Pigeons also can serve as paratenic hosts for Toxocara canis (Galvin 1964).

Various hatching methods have been used to bring out L2 from Toxocara canis resistant infective egg shell. For this purpose, the mechanical hatching methods (Oaks and Kayes 1979; Bowman et al. 1987; Alcântara-Neves and Santos 2008; Smith 1989) and the physiological hatching methods (De Savigny 1975; Ponce-Macotela et al. 2010) have been presented; all of these methods produce the free L2 in the laboratory. Confrontation between the parasite and the host’s immune system is probably essential for the genes coding ES proteins to be expressed; moreover, the above mentioned methods had different results when practiced by different researchers. Therefore we decided to set up a more simple method, in which a real host-parasite interaction occurs. Finally, larval recovery using two in-vitro methods were performed and their results were compared with the alternative in vivo method.

Materials and methods

Preparing infective eggs

Fertilized female worms retrieved from the stool of a naturally infected puppy after treatment with albendazol and then washed many times with PBS. A large number of Toxocara canis eggs were collected directly from the female worm’s uterus and transmitted to 1 % formalin solution. Eggs were cultured 20 days in lab condition, until the infective second stage larvae were observed inside the eggs. In this study we used eggs which were left 6 months in 1 % formalin solution in the refrigerator.

Infecting the pigeons

Certain numbers of eggs were transmitted to separate tubes and washed twice with distilled water to remove formalin. After final centrifuge (4,000 rpm, 2 min) and removing the supernatant, eggs were collected and inserted directly into the pigeon’s crops by means of a simple plastic dropper.

Ten pieces of pigeon were obtained from local sellers across the city Ahvaz, south of Iran; and their feces were examined for any parasitic infestations. Five pigeons were fed 2,000 eggs and the rest were given ~4,000 eggs.

Larvae collection

Pigeons were humanely euthanized on days 1, 2, 3, 4 and 30 post-infection, and their livers and lungs were excised. The mentioned organs were partly minced with scissors and then put into the Baermann’s apparatus. The Baermann’s funnel was filled with 37 °C distilled water containing penicillin (100U/ml), streptomycin (250 μg/ml) and amphotericin B (2 μg/ml). After 8 h incubation in 37 °C, the sediments containing live larvae were collected and larvae were counted under stereomicroscope. Finally, Larvae were filtered through a 20μ nylon mesh to remove the debris.

In-vitro methods

Two in-vitro methods for larvae extraction were also performed

1. Physiological method according to (Ponce-Macotela et al. 2010). Briefly, 4,000 eggs in two groups were de-coated with sodium hypochlorite (6 % free chlorine) and then incubated 30 min at 37 °C in Hanks’ balance saline solution (pH 2.0). Prior to use, the pH was adjusted to 7.4 with sodium bicarbonate and later adjusted to pH 2.0 with 10 N hydrochloride acid to simulate stomach conditions. The reaction between hydrochloride acid and sodium bicarbonate produces CO₂ which triggers hatching of larvae.

2. Mechanical method according to (Alcântara-Neves and Santos 2008); which is a modification of the method described by De Savigny (1975). De-coating performed in the same way at first. In brief, egg hatching was accomplished by incubating them in a bottle containing RPMI and sterile glass beads, and gently shaking by hand for 3–5 min.

Results

All of the pigeons were infected with L2 of Toxocara canis. Results of counting the larvae obtained from the Baermann’s apparatus are summarized in Table 1. Mean percent of larvae recovery from livers and lungs were 36.2 and 0.15 respectively; total 36.3 %. Maximum larvae recovered in day 3 post-infection (55 % total).

Table 1.

Recovery rates of the larvae from livers and lungs of pigeons infected with Toxocara canis infective eggs

No.	Days after infection	No. of fed larvae	Larvae in liver		Larvae in lungs		Total number	Total percent
			No.	Percent	No.	Percent
1	1	2,000	450	22.5	0	0	450	22.5
2	1	4,000	1040	26	0	0	1,040	26
3	2	2,000	1100	55	1	0.01	1,101	55
4	2	4,000	1700	42.5	3	0.07	1,703	42.5
5	3	2,000	780	39	5	0.2	785	39
6	3	4,000	1660	41.5	8	0.2	1,668	41.7
7	4	2,000	700	35	8	0.4	708	35.4
8	4	4,000	1290	32.2	10	0.25	1,300	32.5
9	30	2,000	680	34	5	0.2	685	34.2
10	30	4,000	1380	34.5	9	0.22	1,389	34.7

Considering the fact that only live and active larvae have the ability to pass through the tissues and also the double layered gauze covering the Baermann’s sieve; nearly all the recovered larvae were alive.

9.9 % of the total 4,000 larvae could hatch when exposed to pH changes using Ponce-Macotela et al. (2010) method. Similar result gained in the second in-vitro method and only 6 % of eggs hatched (Table 2).

Table 2.

Hatching rates of Toxocara canis infective eggs by in vitro methods

	Test	No. of eggs	No. of hatched larvae	Percent of hatched larvae	Total percent
Physiological method	1	2,000	210	10.5	9.9	Ponce-Macotela et al. 2010
	2	2,000	189	9.45
Mechanical method	3	2,000	130	6.5	6	Alcântara-Neves and Santos 2008
	4	2,000	113	5.6

Discussion

The second stage larvae of Toxocara canis are the best choice for performing immunodiagnostic and immunoprophylactic tests both in human and animals.

As shown in Table 1, average larvae recovery in the first day after infection (24.2 %) was significantly lower than subsequent days (39 %). The reason is probably that not all of the newly hatched larvae could migrate to liver and also lungs in the first day. Moreover, (Maizels et al. 2000) suggested that the expression of genes coding the E–S antigens requires at least three days keeping larvae in the incubator; therefore the best time for larvae recovery from infected pigeons is probably days 2 and 3 after infection. Larval yield in the liver is approximately constant in the days after first day.

Migration pattern of Toxocara canis L2 in pigeon is described by Galvin (1964); who had traced the larvae by digesting tissues in acid-pepsin and then using the Baermann’s apparatus for retrieving them. According to this study, the maximum numbers of larvae were recovered from the liver and then the lungs. Other organs such as brain and muscles had a few number of larvae or even were free of them. He also showed that the number of larvae recovered from the lungs and livers of infected poultries in the first 24 h post-infection were clearly less than subsequent days; although this difference did not exist in the examined pigeons.

Similar studies with different purposes have also been performed in other paratenic hosts. Most of the larvae recovered (more than 70 %) from mouse existed in skeletal muscles (carcass), according to Tuzer et al. (2002). They found fewer larvae in liver, lungs, brain and other organs. In gerbils, Alba-Hurtado et al. (2009) recovered most of the larvae from lung in days 3–5 post-infection, but after this time, carcass had the most parasite yield compared to other organs. Most of larvae recovered from pigs, existed in lungs (Helwigh et al. 1999). In poultry, results of other studies (Galvin 1964; Gargili et al. 1999) were similar to our results, and they have shown that liver have had the maximum number of larvae.

Results of in-vitro methods were completely disappointing. Ponce-Macotela et al. (2010) reported 96 % egg hatching; but we could retrieve only 10 % of larvae, although we tried to perform the method with all the details. Hatching induction with Alcântara-Neves and Santos (2008) method and after sever shaking could not exceed 6 %, while they claimed that all of the eggs hatched with their method. Most of larvae gained with this method died as a result of shaking. It is important to note that only mRNA of live larvae is suitable for cDNA making. Therefore we could not compare the efficacy of in-vitro methods but it was obvious that the in-vivo method we described is considerably simpler and gives more invariable results in comparison with in-vitro methods. Results of this study show that, by feeding 4,000 larvae to a pigeon, we can expect to recover about 1,500 s stage larvae (36 %) just in the liver. This amount of larvae is enough for successfully performing mRNA extraction procedure; as we extracted adequate amount of mRNA for cDNA making in continue.