Isolation of Toxoplasma gondii from Horse Meat in Egypt
Portions of heart, liver, skeletal and diaphragmatic muscles obtained from 150 slaughtered horses at Giza-Zoo abattoir were used for bioassays in mice and cats. T. gondii tachyzoites were isolated successfully from the peritoneal exudates of the inoculated mice 6-8 days post inoculation with pooled horse tissues. Whereas, T. gondii tissue cysts containing bradyzoites were detected in the impression smears of mice brain on the 45th days or more post infection. The oocysts were detected in feces of cats 3-6 days post feeding on horse tissues containing tissue cysts. The oocysts became sporulated within 3-5 days in 2.5% Potassium dichromate. A total of 79 out of 150 horse meat samples were found to be infected with an incidence rate of 52.6 %. This is the first trial for isolation of T. gondii infective stages from horses in Egypt. Moreover, this study pointed out to the high infection rate of T. gondii in horse meat which may be considered as an important source of infection to wild zoo-animals in Egypt and humans in some countries if consumed raw or insufficiently cooked.
Toxoplasma gondii is a protozoan parasite of warm-blooded animals including man. It has a worldwide distribution. Cats, including all felines, are the definitive hosts which excrete environmentally-resistant oocysts in their feces. Hosts become infected by ingestion of food or drink contaminated with oocysts or by ingesting undercooked meat from infected animals with T. gondii. It causes mental retardation and loss of vision in congenitally-infected children and abortion in pregnant women and livestock (Dubey et al., 2005).
Among the major food animals, viable T. gondii has been found in pork,
mutton, chicken and horse meat (Dubey et al., 1999). Munday (1970), in
Australia, isolated T. gondii from the diaphragm of one of four horses.
AL-Khalidi et al. (1980), in the USA, isolated T. gondii from
tissues (heart, diaphragm, spinal cord, brain, tongue, skeletal muscles, liver
and kidney) of 4 out of 9 ponies experimentally fed infective T. gondii
oocysts by mouse inoculation and feeding of cats. Dubey (1985), recovered T.
gondii from tissues of equids (tongue, intestine, muscles, brain and heart)
when were bioassayed in mice and cats and proved that T. gondii can persist
in edible tissues of living equids up to 476 days. Turner and Savva (1990) reported
the presence of T. gondii DNA in lesions from placenta of a mare in UK.
While evidence that T. gondii causes neurologic disease in horses is
not definite, there are reports of T. gondii infection in the eyes of
aborted foals in UK (Turner and Savva, 1991 and 1992) and in CNS of horses in
Brazil (Macruz et al., 1975).
Although many studies have been reported in many countries on isolation of T. gondii from horse tissues, there is no any previous trial on isolation of T. gondii from horses in Egypt. Therefore, the main objectives of this study are isolation of T. gondii infective stages from meat and tissues of the slaughtered horses by mouse inoculation and cat feeding and also detection the rate of T. gondii infection within horse's tissues in Egypt.
MATERIALS AND METHODS
Isolation of Toxoplasma gondii strain from horse meat
Collection and preparation of meat samples: Meat and tissue samples were
obtained from 150 slaughtered horses at Giza-Zoo abattoir, prepared from heart,
liver, skeletal and diaphragmatic muscles as described by Kotula et al.
(1983); a pooled sample, about 50 to 100 g, from the tissues and organs of the
slaughtered horses, cut into small cubes about 5x5x5 cm and stored at 4°C
for few hours until used for bioassays in mice and cats.
Mice: Swiss albino laboratory breed mice about one month old, their body
weights vary from 25 to 35 g, were kept in clean cages and fed on pelleted food
and water was offered. These mice were used for bioassay of the suspected infected
tissues in order to isolate T. gondii tachyzoites from the peritoneal
cavity or obtaining the T. gondii tissue cysts containing bradyzoites
from their brain. Also, these mice were used to maintain the tachyzoites of
the isolated T. gondii equine strain in the laboratory.
Cats: Three kittens about one month old were isolated from their mothers, kept separately and fed only on boiled or pasteurized milk and bread before using in the experiment. These kittens were used to isolate the T. gondii local strain oocysts. Meanwhile their feces were parasitologically examined daily for 7 days to insure that they were free from any of coccidian infection.
Bioassays of meat and tissues of slaughtered horses
Bioassay in mice: Prepared pooled horse meat samples were digested by pepsin
as the procedures described by Sharma and Dubey (1981). The suspended sediment
of each digested sample was inoculated separately into two groups of mice (each
of 2 mice), one group inoculated intra-peritonealy and the other subcutaneously
(1 mL/mouse). After one week from inoculation, the first group was scarified
and the peritoneal exudates were examined microscopically to identify the tachyzoites.
While after 4-6 weeks, impression smears from brains of the second group were
prepared and examined to detect T. gondii tissue cysts containing bradyzoites.
Bioassay in cats: Each of the three cats was fed on about 250 g of infected pooled horse meat samples, which previously showed T. gondii bradyzoites by pepsin-digestion, in order to isolate T. gondii oocysts as the procedures described by Davis and Dubey (1995). Fecal samples from each cat were collected daily from 3-14 days post experimental feeding and examined parasitologically by concentration floatation technique. The recovered oocysts were mixed with 2.5% Potassium dichromate solution, placed in wide Petri dish at room temperature for 5-7 days to allow the sporulation and then stored at 4°C.
Maintenance of the isolated T. gondii strain: The virulent locally isolated strain of T. gondii obtained from horse meat was maintained in Department of Zoonosis, National Research Center by serial passages in mice. The peritoneal fluid containing about 2x106 tachyzoites was inoculated into 3 to 5 mice (0.2 mL each) and further inoculation was done every 2-4 days according to the method of Johnson et al. (1979).
T. gondii infection rate in horse meat samples: A total of 79 out of 150 (52.6%) of horse meat samples were found to be infected with T. gondii and viable infective stages were detected from mice inoculated intra-peritonealy with the pepsin-digested meat samples.
Isolation of T. gondii infective stages from horse tissues
T. gondii tachyzoites: Viable infective tachyzoites were recovered
from the peritoneal exudates of mice inoculated 6-8 days earlier with infected
digested horse meat samples. The tachyzoites were found inside leucocytes (lymphocytes
and macrophages) or free in the peritoneal exudates after rupture of leucocytes.
The isolated tachyzoites were often crescent in shape or banana shape, pointed
at one end and rounded or blunt at the other one, their sizes ranged from 2-4
μ in width x 5-8 μ in length (mean 3x7 μ) (Fig.
1A and B).
||Giemsa stained T. gondii tachyzoites; A:x400, B:x1000
||Giemsa stained T. gondii tissue cysts; A:x400, B:x1000
||T. gondii oocysts A: Unsporulated, B: Sporulated x
T. gondii tissue cysts with bradyzoites: Tissue cysts of T. gondii were noticed in the impression smears of mice brain or prints on the 45th day or more post infection. They were usually sub-spherical
to spherical in shape and their cyst walls were thin, elastic and well-defined
enclosing crescent shaped bradyzoites. The young tissue cysts may be small (5-10
μ in diameter) contain only 4 to 8 bradyzoites, but the mature older tissue
cysts were 50 to 200 μ in diameter and contain several hundreds of bradyzoites
(Fig. 2A and B).
T. gondii oocysts: T. gondii oocysts were isolated from feces of the three cats 3-6 days post infection with horse tissues containing T. gondii tissue cysts (pre-patent period), these oocysts were still released in cat feces for a period of 10-12 days from the beginning of the release (patent period). The fresh recovered unsporulated oocysts were identified by their spherical shapes, their sizes which ranged from 9-11 μx11-13 μ (mean 10x12 μ) and the embryonic cells which were in close contact to the cyst wall (Fig. 3A). The sporulated oocysts were spherical to ellipsoid in shape and their sizes were ranged from 10-13 μx12-15 μ (mean 11.5x13.5 μ). Each oocyst contains two sporocysts and each contains four sporozoites and sporocystic residual bodies (Fig. 3B).The sporulation of fresh oocysts were observed after 3-5 days under suitable conditions of temperature and oxygenation by using 2.5% Potassium dichromate at room temperature.
T. gondii tachyzoites were isolated successfully from the peritoneal exudates in the inoculated mice 6-8 days post inoculation (DPI) with the digested horse tissue samples. Whereas, T. gondii tissue cysts containing the bradyzoites were detected in mice brain prints after 45 DPI. Such results are in agreement with that of Dubey and Frenkel (1973), who found T. gondii tachyzoites in the impression smears obtained from intestinal lymph nodes on the 6th day post inoculation. However, Dubey and Frenkel (1976) recognized T. gondii tissue cysts in brain prints 3-4 weeks post infection, earlier than the findings obtained during this study. Also, Dubey (1997) detected T. gondii tachyzoites and cysts in the mesenteric lymph nodes, lung and brain after 3, 7 days and 2 months post infection, respectively. The discrepancy between the results of the present study and those recorded by previous authors might be ascribed to the difference in the strain of the parasite, route of inoculation, age of inoculated mice and the development of their immune system.
T. gondii oocysts were detected in feces of cats 3-6 days post feeding on horse tissues containing Toxoplasma tissue cysts and these oocysts were still released in cat feces for a period of 10-12 days. The isolated oocysts were in accordance with that reported by Dubey and Beattie (1988) and Dubey (2001), who declared that cats are very sensitive indicator of T. gondii infection because cats fed even a few T. gondii bradyzoites can shed a million of oocysts. In the present study, T. gondii oocysts ranged between 10-12 μ in case of unsporulated oocysts but the sporulated forms were 11.5-13.5 μ. These were comparatively similar to those measurements of oocysts obtained by Dubey (1998a), who reported that T. gondii oocysts ranged from 9x11 μ for unsporulated oocysts and 12x13 μ for sporulated ones. The oocysts became sporulated within 3-5 days in 2.5% Potassium dichromate solution. In this regard Zaman (1970) and Dubey (1998b and 2002) proved that oocysts sporulation is temperature dependent. Moreover, Dubey (1998a) added that good ventilated media as 2.5% Potassium dichromate or 2% H2SO4 with slight shaking had remarkable effect on the sporulation period of T. gondii oocysts.
In conclusion, this study is the first trial for isolation of T. gondii infective stages from horses in Egypt and detection of the incidence rate of infection among horse meat. It is important, therefore, to evaluate the clinical significance of equine toxoplasmosis and to consider the public health aspects, especially if horse meat infected with toxoplasmosis is to be offered for animal or human consumption in some countries.
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