A novel candidate vaccine for cytauxzoonosis inferred from comparative apicomplexan genomics

Abstract

Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.

Figure 1. Distribution of cytauxzoonosis in the United States.

Figure 2. Life cycle of Cytauxzoon felis.

The acute tissue stage of disease (the schizogenous phase) is characterized by wide spread dissemination of schizonts which form parasitic thrombi throughout the body resulting in a disease course that is typically fatal. Hosts that survive this acute tissue phase develop a chronic yet fairly innocuous erythroparasitemia with merozoite-infected red cells.

Figure 3. Conserved gene synteny between T. parva p67 and C. felis cf76.

cf76 is identified in silico within a highly conserved syntenic block of genes similarly to the leading vaccine candidate for T. parva, p67.

Figure 4. Assessment of feline sero-reactivity to cf76 and cf76 fragments by western blot.

Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with pooled sera (1∶500) from cats surviving C. felis infection (A) or naive cats (B).

Figure 5. Assessment of purified cf76 and cf76 fragments by western blot.

Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with anti-HIS N-terminal tag (A) and anti-HA C-terminal tag antibodies (B).

Figure 6. In situ hybridization to identify transcription of cf76 in C. felis-infected lung tissue.

A. Hematoxylin and eosin stained lung tissue demonstrating schizonts forming a parasitic thrombus within a pulmonary vessel, 20X, B. Negative sense riboprobe, hematoxylin and eosin counterstain, numerous positive cells (brown) are demonstrating intracytoplasmic presence of cf76 antigen, 20X, C. Irrelevant negative sense riboprobe, hematoxylin and eosin counterstain, 20X.