Association of deficiency in antibody response to vaccine and heterogeneity of Ehrlichia risticii strains with Potomac horse fever vaccine failure in horses

Abstract

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), which continues to be an important disease of horses. Commercial inactivated whole-cell vaccines are regularly used for immunization of horses against the disease. However, PHF is occurring in large numbers of horses in spite of vaccination. In a limited study, 43 confirmed cases of PHF occurred between the 1994 and 1996 seasons; of these, 38 (89%) were in horses that had been vaccinated for the respective season, thereby clearly indicating vaccine failure. A field study of horses vaccinated with two PHF vaccines indicated a poor antibody response, as determined by immunofluorescence assay (IFA) titers. In a majority of horses, the final antibody titer ranged between 40 and 1,280, in spite of repeated vaccinations. None of the vaccinated horses developed in vitro neutralizing antibody in their sera. Similarly, one horse experimentally vaccinated three times with one of the vaccines showed a poor antibody response, with final IFA titers between 80 and 160. The horse did not develop in vitro neutralizing antibody or antibody against the 50/85-kDa strain-specific antigen (SSA), which is the protective antigen of the original strain, 25-D, and the variant strain of our laboratory, strain 90-12. Upon challenge infection with the 90-12 strain, the horse showed clinical signs of the disease. The horse developed neutralizing antibody and antibody to the 50/85-kDa SSA following the infection. Studies of the new E. risticii isolates from the field cases indicated that they were heterogeneous among themselves and showed differences from the 25-D and 90-12 strains as determined by IFA reactivity pattern, DNA amplification finger printing profile, and in vitro neutralization activity. Most importantly, the molecular sizes of the SSA of these isolates varied, ranging from 48 to 85 kDa. These studies suggest that the deficiency in the antibody response to the PHF vaccines and the heterogeneity of E. risticii isolates may be associated with the vaccine failure.

FIG. 1

The 50/85-kDa SSA gene-based DAF profile of the new 1994 isolates showing variation in the size of the amplified products. The PCR-amplified products from the 1994 isolates and the 90-12 and 25-D strains of E. risticii were electrophoresed on a 1% agarose gel. Lanes: 1 and 19, DNA molecular size markers; 2, 25-D strain; 3, 94-2; 4, 94-3; 5, 94-24; 6, 94-27; 7, 94-30; 8, 90-12 strain; 9, 94-22; 10, 94-25; 11, 94-29; 12, 94-8; 13, 94-28; 14, 94-31; 15, 94-37; 16, 94-49; 17, 94-50; 18, negative control. The ATCC vaccine strain is not shown due to lack of lanes. However, the molecular size of the DAF product of the strain was verified several times to be 1.75 kb. The tabular data divides the isolates into six groups based on their amplified DNA fragment sizes. Groups representing the three known strains are indicated. The last row of the tabular data indicates the total number of isolates in each group; the percentage representation of the total number of all isolates is shown in parentheses.

FIG. 2

Western blots of new 1994 isolates reacted with the monospecific antisera against the recombinant 85-kDa SSA, showing SSA bands of various sizes ranging from 48 to 85 kDa. Bands are visible for 85-kDa SSA (isolate 94-29 and strain 90-12), 60-kDa SSA (isolates 94-22 and 94-25 [the latter also has an SSA band of 58 kDa]), 58-kDa (isolate SSA 94-31), 50-kDa SSA (isolates 94-2, 94-3, 94-8, 94-24, and 94-27 and strain 25-D and the ATCC vaccine strain), and 48-kDa SSA (isolates 94-28, 94-37, and 94-50). Isolate 94-25 containing 60-kDa SSA was from a mixed culture with an isolate containing 58-kDa SSA. This mixed culture was confirmed by PCR, since amplification of the full-length gene with primers ECP-1 and ECP-2 resulted in two distinct fragments of the corresponding gene sizes. It is important to note that since crude infected cell culture preparations were used, products resulting from proteolytic degradation caused the appearance of multiple bands. In each lane, the major band with the largest molecular size was considered to represent the size of the SSA. To confirm the assigned molecular size of each SSA, the corresponding complete gene was amplified with primers ECP1 and ECP2 (3). The sizes of amplified fragments correlated with the gene sizes calculated on the basis of the assigned molecular size (data not shown).