PREVALENCE OF TOXOPLASMOSIS IN RABBITS IN soum BOHEMIA

Hej Hi!ek K., I. Li ter4k: Prevalence of Toxoplasmosis in Rabbits in South Bohemia. Acta vet. Bmo,63,1994:145-150. The incidence and prevalence of toxoplasmosis in rabbits in small flocks in the district of Strakonice was investigated from 1981 to 1986. A total of 366 slaughter rabbits from 48 small flocks were examined using Sabin-Feldman dye test (DT) and isolation attempts. Toxoplasma gondii antibodies were detected in 194 (53.0%) rabbits and the causative agent was demonstrated in the tissues of 54 (17.8%) out of 304 rabbits in which bio-assay was concluded. Of the 54 slaughter rabbits having T. gondii in the tissues 18 (33.3%) were devoid of antibodies when examined with DT at slaughter. Of the 48 husbandmen 36 supplied 5 or more rabbits. In all these 36 small flocks rabbits with T. gondii antibodies were found and in 23 (63.9%) of them the causative agent was demonstrated. In 4 other small flocks 86 blood serum samples were examined with DT upon repeated blood collections; 62 (72.1 %) of them showed T. gondii antibodies. The incidence of antibodies varied from flock to flock, ranging between 44% and 84%, and the highest titre was 16384. At blood collections repeated several times during one year some rabbits showed marked seroconversion and retained high antibody titres. One rabbit, however, in the same flock had no antibodies or showed them at the titre of 4 at the highest. Toxoplasmosis continues to be a problem in small flocks of rabbits, posing serious hygienic and epidemiological hazards. Slaughter rabbits from small flock are the most T. gondii affected of all common slaughter animal species. Considering the increasing production of rabbit meat due attention to toxoplasmosis in rabbits is required. Rabbit. Toxoplasma gondii, antibody. isollltion, smallfloclcs. Toxoplasmosis in rabbits has received less attention in the world literature than toxoplasmosis in other farm animal species. Some relevant data are to be found in the monograph by D u be yand B e a tt i e (1988). In our country H a v I I k and HUb ne r drew attention to the incidence of toxoplasmosis in rabbits in 1960. Having examined blood sera of 110 rabbits from 10 flocks with Sabin-Feldman dye test (DT) they detected Toxoplasma gondii antibodies in 94.5% of the sera in titres of 4 to 1 048 576. In their isolation attempts they demonstrated the presence of T. gondii in 2 out of 8 rabbits included in the experiment. The prevalence of T. gondii antibody in the sera of rabbits from all 10 flocks was approximately the same. Similar results were later reported by Z4stha et al. (1966) who using DTfound T. gondii antibodies in 91.3% of the sera and drew attention to possible connexion between toxoplasmosis in rabbits and T. gondii infection in man. HUbner and UhHkov4 (1970) using microprecipitation in agar gel detected T. gondii antibodies in the sera of 23.2% of rabbits, Sima and R a§ I n (1973) examining a large sample ofslaughterrabbits with the complement-fixation test (CFl") found T. gondii antibodies in 54.9% of the animals and Luke§ov4 et al. (1984) using DT detected T. gondii antibodies in 86.4% of the rabbits examined. According to the Surveillance of Anthropozoonoses in the Czech Republic in 1980 to 1988 the proportion of rabbits serologically positive for tox,oplasmosis by DT was 42.8% and isolation attempts yielded T. gondii in 11.3% of the rabbits examined. Infection of rabbits with T. gondii is generally not manifested by clinical signs of disease. In some cases, however, clinical manifestations of the disease were described including injury to the CNS, paralysis of hind limbs, abortion, congenital hydrocephalus, together with more general symptoms such as inappetence, emaciation, rhinitis and enteritis (Peeters and Halen 1978, Manfredini 1978, Zardi et al. 1979, Bergmann et al. 1980). The infection is caused by ingestion of feed contaminated with T. gondii oocysts (Peeters and Halen 1978, Ianitschke 1979, Malukiene 1989a). Nevertheless, toxoplasmosis in rabbits may apparentlybe also due to congenital transmission (Uhllkov4 and HUbner 1973, Werner et al. 1977). Rabbits are highly susceptible to T. gondii infection. Differences, however, exist in the degree of infection, depending on husbandry conditions (Sima andRaUn 1973, Peeters etal. 1979) as well as on individual susceptibility (Malukiene 1989b). The present report was designed as part of our comprehensive study on epizootiology of toxoplasmosis in the dismctofStrakonice in the 1980's(Hejlli!ek andLiter4k 1992,1993a, b,Liter4k etal.I992,Litedk and Hejlii!ek 1993) with the objective to assess the incidence ofT. gondii antibodies and of the causative agent in

In 4 other small flocks 86 blood serum samples were examined with DT upon repeated blood collections; 62 (72.1 %) of them showed T. gondii antibodies.The incidence of antibodies varied from flock to flock, ranging between 44% and 84%, and the highest titre was 16384.At blood collections repeated several times during one year some rabbits showed marked seroconversion and retained high antibody titres.One rabbit, however, in the same flock had no antibodies or showed them at the titre of 4 at the highest.
Toxoplasmosis continues to be a problem in small flocks of rabbits, posing serious hygienic and epidemiological hazards.Slaughter rabbits from small flock are the most T. gondiiaffected of all common slaughter animal species.Considering the increasing production of rabbit meat due attention to toxoplasmosis in rabbits is required.Rabbit.Toxoplasma gondii, antibody.isollltion, smallfloclcs.
Toxoplasmosis in rabbits has received less attention in the world literature than toxoplasmosis in other farm animal species.Some relevant data are to be found in the monograph by D u be yand B e a tt i e (1988).
In our country H a v I I k and HUb ne r drew attention to the incidence of toxoplasmosis in rabbits in 1960.Having examined blood sera of 110 rabbits from 10 flocks with Sabin-Feldman dye test (DT) they detected Toxoplasma gondii antibodies in 94.5% of the sera in titres of 4 to 1 048 576.In their isolation attempts they demonstrated the presence of T. gondii in 2 out of 8 rabbits included in the experiment.The prevalence of T. gondii antibody in the sera of rabbits from all 10 flocks was approximately the same.Similar results were later reported by Z4stha et al. (1966) who using DTfound T. gondii antibodies in 91.3% of the sera and drew attention to possible connexion between toxoplasmosis in rabbits and T. gondii infection in man.HUbner and UhHko-v4 (1970) using microprecipitation in agar gel detected T. gondii antibodies in the sera of 23.2% of rabbits, Sima and R a § I n (1973) examining a large sample ofslaughterrabbits with the complement-fixation test (CFl") found T. gondii antibodies in 54.9% of the animals and Luke §ov4 et al. (1984) using DT detected T. gondii antibodies in 86.4% of the rabbits examined.According to the Surveillance of Anthropozoonoses in the Czech Republic in 1980 to 1988 the proportion of rabbits serologically positive for tox,oplasmosis by DT was 42.8% and isolation attempts yielded T. gondii in 11.3% of the rabbits examined.
Infection of rabbits with T. gondii is generally not manifested by clinical signs of disease.In some cases, however, clinical manifestations of the disease were described including injury to the CNS, paralysis of hind limbs, abortion, congenital hydrocephalus, together with more general symptoms such as inappetence, emaciation, rhinitis and enteritis (Peeters and Halen 1978, Manfredini 1978, Zardi et al. 1979, Bergmann et al. 1980).The infection is caused by ingestion of feed contaminated with T. gondii oocysts (Peeters and Halen 1978, Ianitschke 1979, Malukiene 1989a).Nevertheless, toxoplasmosis in rabbits may apparentlybe also due to congenital transmission (Uhllkov4 and HUbner 1973, Werner et al. 1977).
Rabbits are highly susceptible to T. gondii infection.Differences, however, exist in the degree of infection, depending on husbandry conditions (Sima andRaUn 1973, Peeters etal. 1979) as well as on individual susceptibility (Malukiene 1989b).
The present report was designed as part of our comprehensive study on epizootiology of toxoplasmosis in the dismctofStrakonice in the 1980's(Hejlli!ek andLiter4k 1992,1993a, b,Liter4k etal.I992,Litedk and Hejlii!ek 1993) with the objective to assess the incidence ofT.gondii antibodies and of the causative agent in rabbits kept in small flocks under traditional husbandry and feeding practices, compare these results with the incidence of toxoplasmosis in other animal species and evaluate the development in the incidence of toxoplasmosis in rabbit flocks in the area under study from the 1960's to the present time.

Materials and Methods
A total of 366 slaughter rabbits coming from 48 small flocks in the distict of Strakonice in the Czech Republic were examined for toxoplasmosis in September to November 1984.Blood samples and liver, diaphragm and brain specimens were collected at slaughter.The blood sera were examined with Sabin-Feldman dye test (DT) according to the standard method of the Central State Veterinary Institute, Prague, only in the dilution of 1 :4.From the liver, diaphragm and brain specimens of each rabbit a pooled suspension was prepared in buffered saline with antibiotics (600 000 IV peniCillin G and 1 g streptomycin in 1 000 ml saline) and injected intraperitoneally into two toxoplasma-negative mice.Unless the mice died earlier, they were killed and exsanguinated after 5 weeks.Their sera were examined with DT.From their brains compressive preparations were prepared for demonstration of T. gondii cysts.The result of bioassay on mice was regarded as positive if specific antibodies were detected in their blood sera with DT at the titre of 4.
In other 4 small randomly chosen flocks in the same district rabbits were repeatedly subjected to serological examination with DT and to examination with microprecipitation in agar gel (MP A) from 1981 to 1986.MP A was carried out using kits and according to the method of the Institute of Sera and Vaccines, Prague.The examinations were made regularly at 3-months intervals.In DT the final titre level was assessed whenever a sufficient serum volume was available.The initial dilution was 1 :4.In MPA the presence of precipitating antibodies was evaluated only qUalitatively.

Results
A total of 366 slaughter rabbits were examined.Of these 194 (53.0%) exhibited T. gondii antibodies upon examination with DT.Of 304 rabbits in which bio-assay on mice was concluded 54 (17.8%) were found to have T. gondii in the tissue specimens examined.
Of the 54 rabbits in whose tissues the presence of T. gondii was demonstrated by bio-assay 36 (66.6%) had T. gondii antibodies in their sera when examined with DT; the remaining 18 (33.3%)rabbits were without antibodies.
In 36 flocks the examination covered 5 or more rabbits.In all these flocks rabbits with T. gondii antibodies were found and in 23 (63.9%) of these flocks T. gondii were demonstrated by bio-assay.- In 4 other small flocks (Nos.I to N) where rabbits were blood-sampled repeatedly (Table 1) a total of 86 blood serum samples were examined with DT.Of these serum samples 62 (72.1 %) showed T. gondii antibodies in the mean geometric (Xg) titre of 52.The antibodies were found in rabbits of all 4 flocks, even though at varying frequency (44% to 84%) and xg, level (6 to 222).Very high titres were found in flock I and low titres, in flocks IT and Ill.Ot 66 blood serum samples examined with MPA 27 (40,9%) exhibited precipitating antibodies.The difference in the incidence of antibodies detected with DT and MPA was highly significant (X 2 =16.3 df=l P<O.OOI).In some rabbits of flocks I and II blood serum examination was carried out 3 to 7 times during a period of at least 9 months (Table 2).Rabbits Nos. 1 to 5 of flock I retained high antibody titres upon repeated examination and all animals of this flock showed marked seroconversion.In the same flock, however, rabbit No.6 examined during the same period showed either the antibody titre of 4 at the highest or no antibodies at all.Similarly, in rabbit No.7 of flock II the result of SF!' alternated between positive and negative during a period of more than 18 months, the titre of 4 being the highest.

Discussion
Since the spread of toxoplasmosis in rabbit flocks in this country was first reported in the 60' s and its epidemiological impact was suggested, a number of observations on the epizootiology of toxoplasmosis in domestic and free-living animals have been made.In the past 30 years a decrease in the prevalence of toxoplasmosis in slaughter cattle, pigs and domestic fowls was recorded in connexion with the increasing adoption of intensive husbandry practices (Hej licek andLiterak 1992, 1993a, Literak andHej licek 1993).Inrabbits kept in small traditional flocks the situation, however, remains approximately the same as was described byHavlik andHUbner (1960), Zastera etal. (1966) andSima and Rdin (1973).InagreementwithHavlik andHUbner (1960) we regard rabbits as highly susceptible to T. gondii as evidenced by the high frequency of the incidence of antibody and causative agent as well as by high antibody titres.A possible explanation can be seen in the fact that since the 60's the husbandry and feeding practices in rabbit flocks have remained unchanged, i. e. use is made mainly of green forage from areas surrounding the farm buildings to which domestic cats have free access and which apparently are heavily contaminated with T. gondii oocysts from feline faeces.The cruCial role of green forage from oocyst-contamin/lted areas in the transmission of toxoplasmosis to rabbits was demonstrated experimentally by Mal ukiene (1989a).Added to this is possible congenital transmission (Uhlikova andHUbner 1973, Werner et al. 1977) or transmission through milk (Rommel and Breuning 1967) and not even transmission through saliva (Terrag n a et al. 1984) or urine (W i c k ham and C arne 1950) of infected rabbits can be excluded.The differences in the incidence of T. gondii antibodies between the small flocks are, in our view, due to different epizootiological situations and particularly to different degrees of environmental contamination with T. gondii oocysts.
A different epizootiological situation exists in large flocks.Sima and Ra~in (1973)using CFI' found that the proportion of slaughter rabbits with T. gondii antibodies coming from a large flock was 38.4%, with antibody titres over 160 being detected in 0.42% of the animals; in rabbits from small flocks, on the other hand, T. gondii antibodies were found by them in 57.9% of the rabbits, with titres over 160 being detected in 8.1 % of the animals.
Similarly, Pee t e r s et al. (1979) reported that the proportion of serologically-positive rabbits was 22.8% in small traditional flocks of rabbits fed on green forage and only 0.97% in rabbits fed granulated feed under intensive husbandry conditions.
Of interest was the finding of rabbits without T. gondii antibodies or showing only occasionallow antibody titres in flocks heavily contaminated with toxoplasmosis.Similar observations were made in rabbits experimentally infected with T. gondii oocysts by M a I u k i en e (1989b).He explained this finding by differences in the susceptibility of individual animals, which becomes particularly apparent in infections with small numbers of oocysts.
As it is, rabbits kept in small flocks are still the most T. gondii-affected among all the other farm animals, similarly to the findings reported 30 years ago, as became apparent from our studies on toxoplasmosis in cattle, pigs, sheep and domestic fowls in the district of Strakonice (HejIicek and Literak 1992, 1993ab,Literak andHejIicek 1993).
A fact of major importance from the diagnostic point of view in our opinion is the rmding that a third of slaughter rabbits in which T. gondii were demonstrated had no T. gondii antibodies detectable with DT at slaughter.These cases may have represented either recentlyinfected animals in which antibodies had not yet developed or subjects in which antibody response had waned although T. gondii cysts survived in the tissues.
The main hazard of T. gondii transmission from rabbits to man lies in consumption of insufficiently heat-treated tissues and in the handling of rabbits at slaughter if principles of personal hygiene are disregarded.Even T. gondii transmission to attendants cannot be excluded, particularly if the animals are in the acute stage of toxoplasmosis.These hazards should be kept in mind in view of an increasing number of private flocks of considerable size in our country.
Toxoplasmosis in rabbits deserves full epizootiological and hygienic attention.According to the Ministry of Agriculture of the Czech Republic the number of rabbits kept in our country is expected to rise from 13.1 million in 1993 to 14.5 million in 1994.It is estimated that in 1994 a total of 160 000 to 180 000 tons of rabbit meat will come to the market and that 25 000 tons of rabbit meat will be consumed by the husbandmen themselves.Thus the consumption of rabbit meat per person in our country in 1994 is estimated to reach 2.5 kg.

Table I Incidence
of T. gondii antibodies in rabbits from small flocks In the dlstlct of Strakonlce examined for a considerable length 01 time , nslng Sabin-Feldman dye test \ -