English abstract
Theileria lestoquardi the causative agent of malignant ovine theileriosis (MOT) is
widespread in Oman causing massive losses among sheep. The present study examined
the extent of diversity and genetic relatedness of the T. lestoquardi in Oman compared to
that in different geographical areas in Africa (Sudan). The analysis revealed moderate
amount of diversity within both sites Sudan (0.572) and Oman (0.582) and limited
Linkage disequilibrium (LD) between pairs of markers. However, there was a higher
rate of multiplicity of infection (MOI) of T. lestoquardi genotypes per infected animal in
Sudan compared to Oman. Evidence of genetic differentiation between T. lestoquardi
parasites in Sudan and Oman FST (0.295), suggests limited overlap between the parasites
in the two sites.
In view of the high prevalence of mixed species infection of the two ovine Theileria spp
(the pathogenic T. lestoquardi and the less pathogenic T. ovis) in infected sheep, the
present study investigated the dynamics and pattern of within-host interaction between
the two parasites among co-infected sheep and its impact on the disease outcome. qPCR
of T. lestoquardi 18s rRNA and T. ovis 18s rRNA was used to quantify parasites in a
cohort of infected sheep over a period of one year (April 2016 to May 2017). The
prevalence of single pathogenic T. lestoquardi infection was high in the first two months
of the study, associated with a greater parasite density and mortality, compared to mixed
infection. However, in subsequent months, the prevalence of mixed infection increased
gradually to reach 100%, associated with declining clinical symptoms and mortality. T.
lestoquardi density was significantly higher in single infection compared to mixed
infection, indicative of competitive interaction between the two parasites. T. lestoquardi
multiplicity (MOI) increase over time reflecting frequent acquisition of novel genotypes
(super-infection). However, the presence of co-infecting T. ovis did not significantly
influenced the multiplicity of T. lestoquardi genotypes.
The available partial T. lestoquardi genome sequence provided an opportunity to
identify orthologue genes of known transmission blocking antigens in related
apicomplexan parasites. In silico analysis identified nine orthologues of sexual stage
proteins genes of other apicomplexan parasites (Babesia and Plasmodium) in T.
lestoquardi (TL03640, TL14250, TL07240, TL12550, TL09935, TL19820, TL18305,
TL07145 and TL09115) belong to four families; 6-Cys, CPW-WPC, LCCL and Hap2.
The above genes are conserved across the apicomplexan parasites (Plasmodium, Babesia
and Theileria). Sequence analysis of these genes revealed limited, nucleotide and
haplotypes diversity among T. lestoquardi isolates in different regions in Oman. In
contrast, higher nucleotide and haplotypes diversity were seen among the asexual stage
antigen genes (Tlms1 and Tl9). The ratio of nonsynonymous to synonymous substitution
(dN/dS) suggests absence of selection among the sexual stage genes, while, higher
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dN/dS ratio indicative of positive selection was observed for the asexual stage antigen
genes.
Transcriptional analysis of four of the above putative sexual stage protein genes, two 6-
Cys genes (TL03640, TL14250) and two CPW-WPC genes (TL07145, TL18305) were
examined in infected RBCs (piroplasm) and T. lestoquardi cell line (schizonts), infected
sheep and infected ticks. The expression of 6-Cys TL03640, TL07145 and the CPWWPC TL18305 elevated in the infected RBCs, in contrast the 6-Cys gene TL14250
expression was higher in the schizonts stage. Transcripts of TL03640 and TL07145, were
detected in infected ticks but not in the corresponding sheep host. On the contrary, 6-cys
TL14250 and CPW-WPC TL18305 transcripts were detected in both infected ticks and
the corresponding infected sheep. TL18305 showed higher expression in ticks compared
to sheep while the TL14250 exhibited higher expression in sheep compared to ticks.
These results imply that proteins encoded by the TL03640 and TL07145 genes have a
potential role in the sexual phase of the life cycle, and can be considered as a
transmission blocking vaccine.
