ISOLATION OF TOXOPLASMA GONDII IN FREE-LIVING SMALL MAMMALS AND BIRDS

Hejlicek K., F. Prosek, F. Treml: Isolation oj Toxoplasma gondii in Free-living Small Mammals and Birds. Acta vet. Brno, 50, 1981: 233-236. In the period from March to December 1979, 72 specimens of free-living small mammals Jlnd birds were investigated by means of isolation experiments in Toxoplasma-negative white mice. The animals were caught in March, May, July, and November. T. gondii was isolated from their brains. In the respective months T. gondii was isolated in 21.7 %, 10.5 %, 18.7 %, and 18.6 % of the animals. Of the total number of 72 animals the T. gondii isolation was positive in 19.4 %. The causative agent was found in the domestic sparrow (Passer domesticus), field sparrow (Passer montanus), yellow hammer (Emberiza citrinella), domestic mouse (Mus musculus, field mouse (Apodemus sylvaticus) in 17.5 %, 25 %, 20 %, 60 %, and 20 %, resp. The agent was not proved in the redstart (Phoenicurus ochruros), white wagtail (Motacilla alba), house martin (Delichon urbica), serin (Serinus serinus) , goldfinch (Carduelis carduelis). Toxoplasma gondii, occurrence, natural Jocality. Toxoplasmosis is a disease of protozoan etiology rather widely spread in the free nature. The small wild mammals are very important for the maintenance and distribution of the causative agent Toxoplasma gondii in nature (J ira et a1. 1965). It is supposed that the infection is maintained within these species through a permanent intrauterine transmission. Thus the small wild mammals become a reservoir of infection from which T. gondii can spread to other animal species. !stomina et a!. (1973) in the USSR found Sorex araneus, Clethrionomys glareolus, Apodemus sylvaticus, Microtus arvalis, Apodemus fiavicollis to be infested in 21 %, 10 %, 41 %,7.6 %, 100 %, resp. In the USA Eyles et a!. (1959) found that as many as 20 % of the mammals living in the wild had antibodies to T. gondii. In Czechoslovakia, for example, Rosicky et a1. (1969) examined serologically 107 small mammals living in the wild and found these antibodies in 17 % of the animals. Catlir (1972) serologically examined 534 Emall mammals living in the wild and found toxoplasma antibodies in Apodemus fiavicollis, Apodemus sylvaticus and Microtus arvalis in 6.4 %, 3.8 % and 5.8 %, resp. At the same time the author proved T. gondii cysts in two cases after microscopical examination, i. e. in Apodemus fiavicollis and Microtus arvalis. T. gondii occurs rather often also in free-living birds. Haslett and Schneider (1978), for example, serologically examined 563 samples of sera of the starling (Sturnus vulgaris) and proved antibodies in 4.8 %. In Czechoslovakia Catar (1974) found T. gondii antibodies in 40.9 % birds living in the wild. In the present study the authors aimed at isolation of T. gondii from animals living in the wild and caught in the sur.roundings of Emall herds of domestic animals studied. The aim was to verify the occurrence of the agent in the wild animals caught because the domestic animals kept in small herds are in intensive contact with the free nature and it can be anticipated that toxoplasmosis will spread fro m the wild animals also to these herds.

In the period from March to December 1979, 72 specimens of free-living small mammals Jlnd birds were investigated by means of isolation experiments in Toxoplasma-negative white mice.The animals were caught in March, May, July, and November.T. gondii was isolated from their brains.In the respective months T. gondii was isolated in 21.7 %, 10.5 %, 18.7 %, and 18.6 % of the animals.Of the total number of 72 animals the T. gondii isolation was positive in 19.4 %.The causative agent was found in the domestic sparrow (Passer domesticus), field sparrow (Passer montanus), yellow hammer (Emberiza citrinella), domestic mouse (Mus musculus, field mouse (Apodemus sylvaticus) in 17.5 %, 25 %, 20 %, 60 %, and 20 %, resp.The agent was not proved in the redstart (Phoenicurus ochruros), white wagtail (Motacilla alba), house martin (Delichon urbica), serin (Serinus serinus) , goldfinch (Carduelis carduelis).

Toxoplasma gondii, occurrence, natural Jocality.
Toxoplasmosis is a disease of protozoan etiology rather widely spread in the free nature.The small wild mammals are very important for the maintenance and distribution of the causative agent Toxoplasma gondii in nature (J ira et a1. 1965).It is supposed that the infection is maintained within these species through a permanent intrauterine transmission.Thus the small wild mammals become a reservoir of infection from which T. gondii can spread to other animal species.!stomina et a!.(1973) in the USSR found Sorex araneus, Clethrionomys glareolus, Apodemus sylvaticus, Microtus arvalis, Apodemus fiavicollis to be infested in 21 %, 10 %, 41 %,7.6 %, 100 %, resp.In the USA  Eyles et a!.(1959) found that as many as 20 % of the mammals living in the wild had antibodies to T. gondii.In Czechoslovakia, for example, Rosicky et a1. (1969) examined serologically 107 small mammals living in the wild and found these antibodies in 17 % of the animals.Catlir (1972) serologically examined 534 Emall mammals living in the wild and found toxoplasma antibodies in Apodemus fiavicollis, Apodemus sylvaticus and Microtus arvalis in 6.4 %, 3.8 % and 5.8 %, resp.At the same time the author proved T. gondii cysts in two cases after microscopical examination, i. e. in Apodemus fiavicollis and Microtus arvalis.
T. gondii occurs rather often also in free-living birds.Haslett and Schneider (1978), for example, serologically examined 563 samples of sera of the starling (Sturnus vulgaris) and proved antibodies in 4.8 %.In Czechoslovakia Catar (1974) found T. gondii antibodies in 40.9 % birds living in the wild.
In the present study the authors aimed at isolation of T. gondii from animals living in the wild and caught in the sur.roundings of Emall herds of domestic animals studied.The aim was to verify the occurrence of the agent in the wild animals caught because the domestic animals kept in small herds are in intensive contact with the free nature and it can be anticipated that toxoplasmosis will spread fro m the wild animals also to these herds.

Materials and Methods
In the period from March till December 1979 the small wild mammals and birds were caught in 4 intervals from the surroundings of small herds and they were subjected to long-term epizootiological examinations.In this period (from March till December 1979) 72 small animals living in the wild were caught, i. e. 23 in March, 19 in May, 16 in July, 14 in November.For the is~lation of T. gondii in Toxoplasma-negative white mice the brains of the caught animals were employed.The sample was cut into a thick-walled beaker and 15 ml of buffered saline with and addition of antibiotics (STM 1 g, PNC 600 000 I. U. per 1000 ml of saline) was added.Homogenization was performed on a submersile eletric mixer with an exchangeable fitter.One ml of the suspension was applied intraperitoneally to white mice, strain ICR, weigh ing 35 -40 g, delivered as Toxoplasma--negative by the national enterprise Velaz, Prague -Lysolaje.The negativity was serologically confirmed in our labory in a selected specimen of mice delivered.From each specimen 3 mice were inoculated.The mice were constantly observed, after 5 weeks they were bled.From the brain of each infected mouse three compression microscopical preparations were prepared for the demonstration of T. gondii cysts.Mixed samples of blood sera of mice infected with one sample were tested serologically using the Sabin-Feldman reaction (SFR) and microprecipitation in agar gel (MPA).The SFR was performed according to the methods of the USVU (Central State Institute for Veterinary Medicine) in Prague, MPA according to the official methods of the USOL (Institute of Sera and Inoculation Substances).

Discussion
The present results of T. gondii isolation from birds and mammals living in the wild agree with the results of serological examinations performed by other authors in that toxoplasmosis is a considerably widely spread disease in the free nature.Of the total number of animals examined T. gondii was isolated lin 19.4 %.It was found most frequently in animals caught in March (21.7 %) and November (28.6 %).
The authors' results in this respect are similar to the findings of Haslett and Schneider (1978) who proved antibodies to T. gondii during the months of May till November in their serological examinations of starlings.Of the 8 species of wild birds the presence of T. gondii was proved in three.However, it is necessary to bear in mind that of the other five species examined were only a few.If more of these had been examined T. gondii isolation could have been anticipated also in these five species of wild birds.The results of the authors' examinations indicate that small wild animals are an important reservoir of toxoplasmosis in the nature from which the causative agent can spread to populations of domestic animals kept especially in small herds where there is a close contact with the nature.