Babesia equi field isolates cultured from horse blood using a microcentrifuge method.

Babesia equi, a causative agent of equine piroplasmosis, was isolated from horses in the Chaco Province of Argentina, a known piroplasmosis endemic region. Fifteen B. equi field isolates were acquired by culture from 23 actively working horses from 2 ranches. The horses appeared healthy with no clinical signs or histories indicative of equine piroplasmosis. All 23 horses had B. equi-specific antibody activity by the indirect fluorescent antibody test and 18 were also complement fixation test positive for B. equi. Equine erythrocytes were prepared for parasite culture using a microcentrifuge tube method. This method greatly reduces the time involved in cell handling and parasite exposure to ambient conditions. By this method, B. equi cultures can be initiated from very small quantities of blood.

Immunoprophylaxis of Rhodococcus equi pneumonia in foals.

An immunoprophylaxis program for R. equi infection of foals has been established on a number of thoroughbred breeding farms in Argentina over the past 4 years. Nearly 800 mares annually were immunized subcutaneously during the last 2 months of pregnancy with 2-3 doses of a vaccine containing soluble antigens of R. equi, including the virulence associated protein (VapA) and 'equi factors' exoenzymes. The mortality from R. equi pneumonia in the foals from vaccinated dams dropped from an average of 3% in the 5 years before the vaccination program was initiated to an average of 1.2% in the 4 years during which the program was applied (P < 0.02). On 3 farms, an additional 380 foals of vaccinated dams annually over 3 years also received at 25 days of age 600-1200 ml of hyperimmune plasma from donors immunized with this vaccine, and as well at 4 days of age in foals with poor transfer of R. equi antibodies from their dams. The average foal mortality because of R. equi in the 380 foals annually to which hyperimmune plasma was administered dropped from 5.8% on these 3 farms to 0.2% (P < 0.05). Active vaccination of foals of unvaccinated mares on an enzootic farm at 20, 30, and 40 days of age did not protect them from mortality due to R. equi pneumonia. Serology was done by complement fixation and an agar gel immunodiffusion (AGID) tests using antigens prepared in the same manner as the vaccine antigens. The immune responses among hyperimmune plasma donors varied considerably as did the responses of vaccinated mares. Of 1117 serum samples with normal post suckling gammaglobulin levels (> 600 mg%) collected at 2 days of age from foals of vaccinated mares, 36% showed a negative or weak positive AGID reaction, while the remainder had positive to strongly positive reactions.

Antigenic analysis of Rhodococcus equi preparations using different horse sera.

An R. equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R. equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R. equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX). The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated with the R. equi vaccine), passively and actively immunized foals' sera, asymptomatic but serologically positive foals' sera sera from R. equi pneumonic foals, an equine APTX antiserum, and a VapA monoclonal antibody (Mab). The vaccine under study had many proteins in high concentrations. Hyperimmune sera reacted strongly with vaccine antigens in the high molecular weight regions. In the low molecular weight range, it reacted in the 14 and less kDa zone. Sera from passively immunized foals reacted similarly but not so strongly. Actively immunized foals gave very weak reactions. With the APTX extract, the Mab reacted with bands at 15-17, 44 and 66 kDa; it reacted weakly with the whole cell and not with the VapA-negative preparations. The APTX antiserum and the Mab reacted strongly with the vaccine at the 14 and less kDa zone, and also with bands at 21, 44 and 66 kDa and very tenuously at 18 kDa, but not in the expected 15-17 kDa zone, suggesting that the native form of VapA is altered but without loss of antigenicity in the vaccine preparation. Our results suggest that other higher molecular weight antigens, in addition to VapA, may be important in inducing antibodies that protect young foals from R. equi pneumonia. These antigens are in high concentrations and in an immunogenic form in the vaccine.