Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes

Abstract

Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus) and sable ( H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.

Theileriosis in naturally infected roan antelope (Hippotragus equinus)

“…16 Furthermore, this assay could detect ILP antigens in tissues from diverse wild artiodactyls and did not cross-react with potentially confusing nontheilerial organisms or leukocytes in lesions that resemble theileriosis histologically. 16 For immunophenotypic characterization of the leukocyte infiltrates, we used commercially available antibodies to detect the following leukocyte antigens: CD20, CD3, MAC387, IBA-1, and CD204. Details of the primary antibodies used, their target cells, optimal heat-induced epitope retrieval (HIER) methods, antibody dilutions and incubation times, chromogens used, and positive tissue controls are summarized in Table 1.…”

“…Staining for ILP antigens required some extra steps. 16 Before the application of the anti- Theileria sp. (sable) antibody B, tissues were treated with a 2% solution of skimmed milk powder in distilled water for 15 minutes at RT in order to block nonspecific background staining.…”

“… Abbreviations: DAB, 3,3′-diaminobenzidine tetrahydrochloride; DPS, Department of Paraclinical Sciences; EDTA, ethylenediaminetetraacetic acid; FVS, Faculty of Veterinary Science; HRP, horseradish peroxidase; IHC, immunohistochemistry; MW, microwave heating; UP, University of Pretoria. a One of 3 serum-purified roan antibodies (A, B, and D) used in a chromogen-based indirect IHC technique that consistently detected leukocyte-associated theilerial antigens in roan and sable tissues. 16 …”

“…(sable) lymph node-derived subcultures in western blots, and in FFPE tissues from infected antelope using IHC. 16 Here, we used antibody B, 1 of 3 purified roan antibodies that consistently detected leukocyte-associated theilerial antigens in roan and sable tissues. 16 Furthermore, this assay could detect ILP antigens in tissues from diverse wild artiodactyls and did not cross-react with potentially confusing nontheilerial organisms or leukocytes in lesions that resemble theileriosis histologically.…”

“…16 Here, we used antibody B, 1 of 3 purified roan antibodies that consistently detected leukocyte-associated theilerial antigens in roan and sable tissues. 16 Furthermore, this assay could detect ILP antigens in tissues from diverse wild artiodactyls and did not cross-react with potentially confusing nontheilerial organisms or leukocytes in lesions that resemble theileriosis histologically. 16 For immunophenotypic characterization of the leukocyte infiltrates, we used commercially available antibodies to detect the following leukocyte antigens: CD20, CD3, MAC387, IBA-1, and CD204.…”