[Immunobiological and antigenic studies of Babesia ovis strains in Bulgaria]. / Imunobiologichni i antigenni prouchvaniia na shtamove Babesia ovis v Bulgariia.

A comparative study was carried out on the immunologic and antigenic properties of 8 strains of Babesia ovis isolated in 5 districts of the country. The immunologic capacity was tested through challenging with each of the strains in group of 4 weaned lambs that were preliminary vaccinated with one of the strains - Veliko Turnovo. Two nonvaccinated animals were included in each group, serving as controls. The strength of immunity was checked by both the temperature and the parasitic reaction as well as through the hematocrit and hemoglobin levels of the challenged lambs. With the exception of one spleen - ectomized animal in each group the vaccinated weaned lambs showed solid immunity against both the homogeneous and heterologic strains. The same result was confirmed also with an analogous biologic experiment with a total of 19870 sheep in 28 enzootic foci in the same 5 districts and in 2 other districts of the country. Cross serologic investigations by means of the complement - fixation test, the agar gel precipitation reaction and IRPA with homologous and heterologous antisera revealed identic or close antigenic properties in the tested strains. In is concluded that the Veliko Turnovo strain can be used to prepare a vaccine for the immunoprophylaxis of babesiasis in sheep in the investigated districts. In is also stated that the serologic reactions, and, more specifically, IRPA can be employed in the antigenic study and differentiation of individual Babesia ovis strains.

[Soluble Babesia ovis antigen]. / Prouchvane vurkhu raztvorim antigen Babesia ovis.

Dissolved antigen of plasma and of erythrocytic cytoplasma was obtained from 4 splenectomized sheep experimentally infected by Babesia ovis. Blood was taken in the stage of acute parasitemia with 23--25% parasite erythrocytes. The antigen was isolated by precipitating the plasma and the erythrocytic cytoplasma three times with an ammonium sulfate solution saturated 50 and 75%. The fraction obtained following dialysis was concentrated after McErlean's method. The specificity and activity of the isolated soluble antigen were proven by RAGP against serums of 6 weaned lamb groups: I group -- with 4 cm3 plasma, II group -- with 8 cm3 plasma, III group -- with 2 cm3 erythrocytic cytoplasma, IV group -- with live vaccine, V group -- with normal plasma and VI group -- normal weaned lambs. The same groups were used to assess the immunogenic properties of the dissolved antigen. Following provocation with a virulent B. ovis strain the lambs of group II proved as resistant as these immunized with live vaccine. The result was confirmed under production conditions -- not one of the 360 lambs immunized with 5 and 10 cm3 plasma became infected, while of the 250 control lambs 30 were infected. The conclusion is drawn that plasma in the stage of acute parasitemia can be used for immunoprophylaxis, but more supplementary studies are needed.

[Production of a Babesia ovis extract and its testing for antigenic properties]. / Poluchavane na ekstrakt ot Babesia ovis i izpitvane na antigennite mu svoistva.

An extract of the parasitic erythrocyte stroma sediment of Babesia ovis was produced. Parasite infestation of the erythrocytes was from 23 to 25%. The sediment was destroyed by three methods: 1) By ultrasound; 2) by twenty-fold freezing and unfreezing in liquid nitrogen; 3) by lyophylization. The supernatant of the destroyed sediment, obtained by ultracentrifugation of 103,5000 g per 1 h was tested as antigen in RCB, RAGP, RIHA. Positive result was obtained for all three reactions with homologous antiserums of lambs immunized with live vaccine and plasmic antigen. In case heterologic anti A. ovis serum and normal sheep serum were used the result was negative. Compared to the "standard" corpuscular antigen for RCB and to the plasmic antigen the extract proved more active.

[Deep freezing of Babesia ovis in liquid nitrogen]. / Dulboko zamraziavane na Babesia ovis v techen azot.

Comparative investigations were carried out to determine the optimal concentration of glycerin and dimethylsulfoxide in deep freezing of sheep erythrocytes infected with Babesia ovis. The blood was obtained from experimentally infected weaned lambs in the phase of acute parasitemia. Equal blood samples, mixed with the same volume of 2-, 3-, 4-, and 5-molar concentrations of the two cryoprotective agents as well as blood samples without such agents were frozen by the slow and the fast method up to--75 degrees C and kept in liquid nitrogen. The cryoprotective effect was evaluated by the hematocrit level of the samples of various concentrations in comparison with samples having no cryoprotective agent, frozen under one and the same conditions and by biologic experiments with susceptible weaned lambs. It was found that 4-molar concentrations of glycerin and dimethylsulfoxide produce an optimal cryoprotective effect, which, with the slow method of freezing was 64 per cent for glycerin and 60.7 per cent for dimethylsulfoxide. Blood frozen under such conditions and stored up to 6 months in liquid nitrogen retained its virulence.

[Trial of a live vaccine against babesiasis in sheep]. / Izpitvane na zhiva vaksina sreshtu babeziozata po ovtsete.

A flock of 86 weaned lambs was used in field conditions to test the vaccination against babesiasis in sheep, using blood containing up to 5 per cent Babesia ovis-infected erythrocytes, referred to as a "live vaccine". The blood was obtained through successive passages performed with the vaccinal strain in four spleenectomized lambs with the parallel treatment of the latter with tetracyclin. The application of the vaccine at rates of 1.75 X 10(7); 3.5 X 10(7); and 7 X 10(7) Babesia organisms was well tolerated, the animals responding with a temperature reaction only. The immunity acquired proved sufficiently stable against the experimental challenge with a triple dose of the maximal vaccinal one as well as against the natural infection to which the flock was exposed within the period between the third and sixth month following vaccination. At the same time babesiasis in an enzootic form developed in the control flock, the test flock showing no cases of diseased animals. The complement-fixing antibodies established from the tenth up to the 90th day after the vaccination was carried out served as an index of the immunity status. The optimal vaccination dose is suggested to be that containing 3.5 X 10(7) Babesiae.

[Immunofluorescent test in the diagnosis of babesiasis]. / Immunofluorestsentnata reaktsiia v diagnostikata na babeziozata

The direct and the indirect immunofluorescence methods were tested in the diagnosis of babesiasis in sheep. The antigen used was material of erythrocyte smears with Babesia ovis, and the conjugate employed in the direct method was obtained from a hyperimmune anti-Babesia ovis serum labelled with fluorescein-isothiocyanate. For the indirect methods rabbit anti-sheep globulin was made of, labelled with the same fluorochrome. The specificity of the direct methods was demonstrated by the absence of fluorescence in the control smears with Th. ovis, A. ovis and E. ovis at a 1:20 dilution of the conjugate (and even in higher dilutions), while that of the indirect methods--by the absence of fluorescence with the control negative serum and the heterologous antisera at the same values of dilution. The comparative testing of the indirect immunofluorescence methods and the complement-fixation reaction in the periodic investigation of 10 lambs (experimentally infected) and 10 sheep (naturally infected) with Babesia ovis revealed that the indirect methods were of higher infected) with Babesia ovis revealed that the indirect methods were of higher sensitivity. This, and the easier production of the antigen gave grounds to assume that indirect methods are more readily applicable in the laboratory diagnostic practice. On the other hand, direct methods are more accurate in the species differentiation of the Babesia agents.

[Occurrence and species of Coccidia in the district of Velikot'rnovski]. / Razprostranenie i vidove koktsidii po govedata v'v Velikot'rnovski okr'g

The study of a total of 1444 fecal samples from calves aged 20 to 90 days and 8 cadavers of coccidiosis-affected calves on 50 dairy farms on the territory of the district revealed coccidiosis on 46 of the farms (92 per cent) and stationary coccidiosis on 13 of them (26 per cent). The percentage of the animals involved on the farms where the disease was stationary was from 50 to 80, and on individual farms it reached up to 100. The parasite burden with the calves showing the acute form of coccidiosis accounted for 20,000 to 32,000 oocysts; calves showing chronical course had 5,000 to 15,000, and the clinically normal calves in the stationary foci had up to 1,000 oocysts per gram of fecal mass. Eight species of coccidia, unknown so far to this country, are described: E. elipsoidalis, E. cilindrika, E. alabamensis, E. auburnensis, E. canadensis, E. Wyomingensis, E. subspherika, and Isospora. The localization of most of these species was determined.