Susceptibility of the domestic duck (Anas platyrhynchos) to experimental infection with Toxoplasma gondii oocysts

Abstract

A total of 28 domestic ducks were divided into seven groups of four ducks. Six groups were inoculated per os with 10¹, 10², 10³, 10⁴, 10⁵ and 10^5.7 oocysts Toxoplasma gondii oocysts (K21 strain, which is avirulent for mice), and the remaining group was used as a control. Antibodies to T. gondii were detected in all ducks by the indirect fluorescence antibody test first on day 7 post-inoculation (p.i.). Antibody titres were found in the range of 1:20 to 1:640 depending on the infectious dose of the oocysts. From day 14 p.i. antibody titres increased to 1:80 to 1:20 480. Between days 14 and 28 p.i. (end of the experiment), antibody titres decreased in 14 ducks, remained the same in seven ducks, and continued to increase in three ducks. Bioassay in mice revealed T. gondii in the breast and leg muscles and the heart (100%, n=47), brain (91%, n=22), liver (54%, n=13) and stomach (46%, n=24). The infected ducks showed no clinical signs; however, the results of bioassay indicate that, compared with some gallinaceous birds, domestic ducks were relatively susceptible to T. gondii infection.

Résumé

Sensibilité du canard domestique (Anas platyrhynchos) à une infection expérimentale par des oocystes de Toxoplasma gondii

Vingt huit canards domestiques ont été divisés en 7 groupes de quatre canards. Six groupes ont été inoculés per os avec 10¹, 10², 10³, 10⁴, 10⁵ et 10^5.7 oocystes de Toxoplasma gondii (souche K21, avirulente pour la souris), un groupe a servi de témoin. Les anticorps vis à vis de T. gondii ont été détectés chez tous les canards par un test d'immunofluorescence indirecte (IFAT) à partir du 7^ème jour après inoculation (p.i.). Les titres des anticorps ont varié du 1/20 au 1/640 en fonction de la dose d'oocystes. A partir du 14^ème jour p.i. les titres des anticorps ont augmenté pour atteindre 1/80 à 1/20480. Entre le 14^ème et le 18^ème jours p.i. (fin de l'expérience) les titres des anticorps ont diminué chez 14 canards, sont restés au même niveau pour 7 canards et ont continué à augmenter pour 3 canards. Un bio essai réalisé chez la souris a révélé que T. gondii était présent dans les muscles de pattes et de la poitrine, ainsi que dans le cœur (100%, n=47), dans le cerveau (91%, n=22), dans le foie (54%, n=13) et dans l'estomac (46%, n=24). Les canards infectés n'ont pas montré de symptômes, cependant, les résultats du bio essai montre que, comparé aux gallinacés, le canard domestique est relativement sensible à l'infection à T. gondii.

Zusammenfassung

Empfänglichkeit der Hausente ( Anas platyrhynchos ) für experimentelle Infektionen mit Toxoplasma gondii -Oozyten

Insgesamt 28 Hausenten (Anas platyrhynchos) wurden in 7 Gruppen zu jeweils vier Enten eingeteilt. Sechs Gruppen wurden per os mit 10¹, 10², 10³, 10⁴, 10⁵ oder 10^5,7 Oozysten von Toxoplasma gondii (K21, avirulent für Mäuse) inokuliert, während die siebte Gruppe als Kontrollgruppe diente. 7 Tage post inoculationem (p.i.) wurden in allen Enten mittels des indirekten Fluoreszenzantikörpertests (IFAT) Antikörper gegen T. gondii nachgewiesen. Die festgestellten Antikörpertiter lagen abhängig von der Oozysteninfektionsdosis zwischen 1:20 und 1:640. Zwischen dem 14. und 28. Tag p.i. (Versuchsende) sanken die Antikörpertiter bei 14 Enten, blieben auf gleicher Höhe bei 7 Enten und stiegen bei drei Enten an. Im Bioassay in Mäusen wurde T. gondii in Brust- und Beinmuskeln sowie Herz (100%, n=47), in Gehirn (91%, n=22), Leber (54%, n=13) und Magen (46%, n=24) entdeckt. Die infizierten Enten zeigten keine klinischen Symptome. Die Ergebnisse des Bioassay weisen jedoch darauf hin, dass im Vergleich mit einigen hühnerartigen Vögeln Hausenten relativ empfänglich für eine T. gondii-Infektion sind.

Resumen

Susceptibilidad del pato doméstico (Anas platyrhynchos) a la infección experimental con ooquistes de Toxoplasma gondii

Un total de 28 patos domésticos se dividieron en 7 grupos de cuatro patos. Seis grupos se inocularon per os con 10¹, 10², 10³, 10⁴, 10⁵ y 10^5,7 ooquistes de Toxoplasma gondii (cepa K21, que es apatógena para el ratón), y el grupo restante se usó como control. Se detectaron anticuerpos contra T. gondii en todos los patos mediante un prueba de anticuerpos de fluorescencia indirecta (IFAT) por primera vez a los 7 días post inoculación (p.i.). Los títulos de anticuerpos se encontraban en un rango entre 1:20–640 en función de la dosis infectiva de ooquistes. Los títulos de anticuerpos incrementaron hasta 1:80–20,480 a partir del día 14 p.i.. Entre los días 14 y 28 p.i. (final del experimento), los títulos de anticuerpos descendieron en 14 patos, se mantuvieron igual en 7 patos, y siguieron aumentando en 3 patos. Un bioensayo en ratón reveló la presencia de T. gondii en los músculos de las patas y pectorales y en el corazón (100%, n=47), encéfalo (91%, n=22), hígado (54%, n=13) y estómago (46%, n=24). Los patos infectados no mostraron signos clínicos, sin embargo, los resultados del bioensayo indicaron que, en comparación con las aves gallináceas, los patos domésticos son relativamente susceptibles a la infección por T. gondii.

Introduction

Toxoplasmosis is a parasitic disease, which may cause serious health problems in animals and humans. Main sources of infection consist of oocysts of Toxoplasma gondii shed by the cat and mammalian and avian tissues, which may contain tissue cysts of T. gondii (Dubey & Beattie, 1988). Little attention has been so far paid to susceptibility of the domestic duck and other birds of the family Anatidae to the infection with T. gondii. The results of earlier seroprevalence studies are currently not considered reliable, in particular if the analysis was based on Sabin–Feldman reaction, which is useless for the detection of antibodies in numerous avian species (Dubey, 2002). Recently, 48 ducks were examined in Egypt by modified agglutination test with 50% positive results (El-Massry et al., 2000). Burridge et al. (1979) examined 16 wild wood ducks (Aix sponsa) in the US by indirect hemagglutination test, and obtained 6% positive results.

Pak (1976) carried out isolation experiments in wild ducks in Kazakhstan. The author examined 57 pintails (Anas acuta) with 1.8% positive results and 93 gadwalls (Anas strepera) with 1.1% positive results. Literák et al. (1992) carried out isolation experiments in wild ducks at five places in the Czech Republic. Out of 184 mallards (Anas platyrhynchos), 22 birds were found positive (12%). In other species the following numbers of positive cases were detected: one of eight (12.5%) in the pochard (Aythya farina), and seven of 25 (28%) in the tufted duck (Aythya fuligula). Literák & Hejlíček (1993) carried out an isolation experiment in 60 domestic ducks. Only one bird (1.7%) was found positive. A case of spontaneous clinical toxoplasmosis in the domestic duck was reported from Argentina, where 50% of 15-day-old to 20-day-old ducklings in a flock died with clinical manifestations of somnolence, corneal lesions and paresis of legs (Boehringer et al., 1962). Moreover, hepatic necrosis was also diagnosed in this case.

The aim of the present study was to use experimental infection of the domestic duck with oocysts of T. gondii K21 strain for the determination of susceptibility of this avian species to toxoplasmosis and for the assessment of distribution of T. gondii tissue cysts in different tissues of infected ducks.

Materials and methods

T. gondii strain

T. gondii oocysts were obtained from faeces of an experimentally infected domestic cat. A serologically negative cat was orally infected with tissue cysts of T. gondii K21 strain, which was obtained from oocysts of a domestic cat in Brno in 1995 and classified among avirulent strains on the basis of biologic and genotypic characteristics (Literák et al., 1998); that is, 100% lethal dose requiring>10³ tachyzoites (Sibley & Boothroyd, 1992). Examination of faeces in the infected cat was carried out according to the methods described by Dubey & Beattie (1988). Sporulation of oocysts took place for 7 days at room temperature in 2% H₂SO₄ and was under continuous microscopic monitoring.

Virulence of sporulated oocysts was tested by experimental infection of Toxoplasma-negative laboratory mice of strain CD-1 (Anlab, Charles River, Prague, Czech Republic) weighing 20 to 22 g. Oocysts, which had been stored in 2% H₂SO₄, were centrifuged and subsequently neutralized with 3.3% NaOH. After another centrifugation and dilution with physiological saline, the infectious doses were prepared containing 10⁰ to 10⁵ oocysts in 50 μl inoculum. In total, 60 mice were inoculated. The mice were divided into six groups of 10 animals, which orally obtained the doses of 10⁰, 10¹, 10², 10³, 10⁴ and 10⁵ oocysts using a micropipette. The cases of deaths were recorded, and 30 days post-inoculation (p.i.) all mice were killed and serologically examined by the indirect immunofluorescence reaction (IFAT).

IFAT was carried out using Sevatest Toxoplasma antigen IFR (Sevapharma, Prague, Czech Republic) and anti-mouse immunoglobulin SwAM/FITC (Sevapharma). The initial serum dilution was 1:20, and titres≥1:20 were considered positive.

Experimental design

In total, 28 White Pekin RITO ducks were used for the experiment. The average weight of each bird was 2.5 kg. The age of the ducks was 50 days. The ducks were divided into seven groups of four birds. Using a micropipette, six groups were orally infected with T. gondii oocysts, and the remaining group was used as a control. The oocysts for inoculation were diluted in physiological saline to obtain the doses of 10¹, 10², 10³, 10⁴, 10⁵ and 10^5.7 oocysts in 0.5 ml inoculum. Prior to the experimental inoculation and on days 7, 14, 21 and 28 p.i., all birds were examined serologically using the IFAT for presence of antibodies to T. gondii. The health status and mortality of the birds were visually inspected for 28 days. The all ducks were killed on day 28 p.i., and their tissues were examined for presence of T. gondii using the isolation assay.

Serology

In the IFAT, commercially available Sevatest Toxoplasma antigen IFR (Sevapharma) and fluorescein-labelled goat anti-duck immunoglobulin G (H+L; Kirkegaard & Perry Laboratoires, Gaithersburg, MD, USA) were used. The sera were diluted in a two-fold series starting at 1:20 as the basic dilution. The titre≥1:20 was considered positive.

Isolation assay

The liver, brain, stomach, heart and muscles (breast and leg muscles together) were homogenized (each organ separately) in 2 ml physiological saline with 600 000 iu penicillin (Biotika n.p., Slovenská Lupča, Slovakia) per 1 l. Two Toxoplasma-negative laboratory mice of strain CD-1 (Anlab), weighing 20 to 22 g, were inoculated intraperitoneally with 1 ml tissue suspension. The cases of death were recorded, and at 30 days p.i. all mice were killed and serologically examined for antibodies against T. gondii (IFAT). The duck tissue was considered positive in cases when antibodies against T. gondii were found in at least one of two inoculated mice.

Results

The virulence of T. gondii K21 strain was tested in mice. The mice inoculated with the doses of 10⁰ to 10⁴ oocysts did not die. Deaths were recorded only in the group with the dose of 10⁵ oocysts, where six of 10 inoculated mice (60%) died on days 7 to 9 p.i. Antibody titres were detected in 60% of the mice (6/10) with the dose of 10⁰ oocyst, 50% (5/10) with 10¹ oocysts, 90% (5/10) with 10² oocysts, 100% (10/10) with 10³ oocysts, 100% (10/10) with 10⁴ oocysts, and 100% (4/4) with 10⁵ oocysts. Because doses>10³ were needed to cause 100% mortality in mice (Sibley & Boothroyd, 1992), the strain was confirmed as avirulent as reported previously by this laboratory (Literák et al., 1998).

Infected ducks did not show any clinical signs of infection. Pathological lesions were not observed at the time of tissue sample collection. Prior to the infection, no antibodies were found in the ducks. On day 7 p.i., antibodies were demonstrated in all the infected ducks. Antibody titres were found in the range of 1:20 to 1:640 depending on the infectious dose of the oocysts. On days 14, 21 and 28 p.i., all infected ducks were serologically positive. The seroconversion in ducks depended to a great extent on the number of T. gondii oocysts in an inoculation dose. The highest titre was 1:20 480, and was observed on day 14 p.i. in three ducks infected with 10⁴ and 10^5.7 oocysts. Between days 14 and 28 p.i. (end of the experiment), antibody titres decreased in 14 ducks, remained the same in seven ducks, and continued to increase in three ducks. Table 1 presents the mean antibody titre with standard deviation for each oocyst dose group.

Table 1. Dynamics of antibody production as measured by indirect fluorescence antibody test in the ducks experimentally inoculated with T. gondii oocysts

		Antibody titres
Bird numbers	Dose of oocysts	7 d.p.i.^a	14 d.p.i.	21 d.p.i.	28 d.p.i.
1 to 4	10^1	1:20±0	1:2880±1395	1:2240±1663	1:2080±831
5 to 8	10^2	1:35±26	1:3540±3966	1:2740±2388	1:2100±1971
9 to 12	10^3	1:200±69	1:1920±640	1:1600±554	1:3200±1920
13 to 16	10^4	1:240±80	1:10 880±6368	1:2560±0	1:3200±1109
17 to 20	10^5	1:480±160	1:8320±3326	1:2560±0	1:2560±0
21 to 24	5×10^5	1:560±139	1:11 520±8960	1:2080±831	1:1920±640
^a d.p.i., day post-inoculation.

During 24 to 48 h after experimental inoculation of mice with duck tissue samples, there were 14 deaths of both inoculated mice (11 cases after inoculation with liver tissue, one case with heart tissue and two cases with brain tissue). Toxoplasmosis was not the cause of death in these cases, but was the result of either Proteus mirabilis, Escherichia coli or a direct toxic effect from the liver homogenate. One or both of the bacteria were isolated from duck liver tissues. The direct toxic effect is possible since all control duck liver samples also caused mortality in inoculated mice. Based on serological examination of the surviving mice, it was established that the level of T. gondii infection was highest in muscles (100%, 24 positive of 24 inoculated mice) and the heart (100%, 23 positive of 23 inoculated mice), followed by the brain (91%, 20 positive of 22 inoculated mice), the liver (54%, seven positive of 13 inoculated mice), and the stomach (46%, 11 positive of 24 inoculated mice) (Table 2).

Table 2. Results of bioassay of organs of experimentally inoculated ducks killed on day 28 p.i.

Dose of oocysts	Muscles	Heart	Brain	Liver	Stomach
10^1	4/4^a	3/3	3/4	0/1	1/4
10^2	4/4	4/4	4/4	1/4	3/4
10^3	4/4	4/4	3/3	2/3	1/4
10^4	4/4	4/4	4/4	1/2	2/4
10^5	4/4	4/4	3/3	1/1	2/4
10^5.7	4/4	4/4	3/4	2/2	2/4
Total	24/24	23/23	20/22	7/13	11/24
^a Data presented as the ratio of the number of positive birds/number of totally tested birds.

Discussion

The K21 strain was classified among avirulent strains on the basis of virulence testing in mice. This result corresponds to previous similar testing of virulence of this strain in mice, when only a dose as high as 10⁵ oocysts was found lethal for 100% of mice (Bártová et al., 2003). Within the framework of genotype characterization of different strains from the Czech Republic, the genotype of the K21 strain was typical for avirulent strains (Literák et al., 1998).

None of the ducks infected with the dose of 10¹ to 10^5.7 oocysts of T. gondii K21 strain died or manifested clinical signs of toxoplasmosis. Nevertheless, on the basis of the results of serological examinations in the ducks and isolation assay, it can be concluded that the domestic duck is a species relatively susceptible to T. gondii infection.

The susceptibility of ducks to the T. gondii infection can be compared with susceptibility in gallinaceous birds, where similar experimental infections with T. gondii were carried out. Oral inoculation with the doses of 10³ and 10⁵ T. gondii oocysts was carried out in the partridge (Perdix perdix), the chukar (Alectoris chukar), the wild guinea fowl (Numida meleagris), the wild turkey (Meleagris gallopavo) and the chicken (Gallus domesticus) (Sedlák et al., 2000). Based on comparison of mortality, seroconversion, titre values and distribution of T. gondii tissue cysts in the bodies of infected birds, it can be concluded that the susceptibility in ducks is most similar to the susceptibility of chukars.

The susceptibility of the domestic duck, however, cannot be compared with the susceptibility of the rock partridge (Alectoris graeca), which was proven to be very susceptible to T. gondii infection in contrast to the chukar (Dubey et al., 1995). Lower susceptibility was found in the pheasant (Phasainus colchicus) and the domestic turkey. The parameters in these species were similar to those in the domestic duck (Dubey et al., 1994). Out of 14 turkeys inoculated with the doses of 10⁵ and 10⁴ oocysts of Me-49 strain only three birds died but the cause of death was reported to not be from the T. gondii infection (Dubey et al., 1993). Wolfson (1941), Simič et al. (1963) and Pak (1965a,b) carried out experimental infection in the domestic duck. Intravenous, intraperitoneal and subcutaneous administration of tachyzoites in ducks resulted in temporary increase of temperature. In ducklings, additional signs reported included a lack of appetite, weakness and death. Oral administration of tachyzoites or tissue cysts led to manifestation of clinical signs only in the ducklings 2 to 3 days old. The ducks older than 14 or 20 days survived without exhibiting clinical signs. Isolation of T. gondii from the tissues on day 14 p.i. was successful. Simič et al. (1967a,b) intravenously administered 10^7.5 to 10^7.8 tachyzoites to laying ducks. The laying was discontinued and resumed after 30 days. No T. gondii was isolated from 37 eggs examined.

The results of the present work demonstrate that the domestic duck is relatively susceptible to the infection with T. gondii. The level of susceptibility of this species is comparable with the chukar. Although clinical signs of T. gondii infection need not develop in cases of avirulent T. gondii strains, tissue cysts may still be present in different parts of the body of the ducks. From the aspect of food safety, it has to be considered that the most intensively infected tissues are skeletal muscles and the heart.

Acknowledgments

The study was funded by Grant No. 161 700 001 (Ministry of Education, Youth and Sports of the Czech Republic).