Both intravenous (IV) and subcutaneous (SC) inoculation of bovine blood infected with T.orientalis buffeli produced parasitosis detectable by PCR within 4 weeks, consistent with previous reports (Hammer et al., 2016; Gibson, 2017). In each case the parasitosis appeared to peak around 5–8 weeks before stabilising at around 2000–10000 GC/ul. It was also noted when H.bancrofti ticks failed to adequately feed and died, that H.longicornis nymphs failed to transmit T.orientalis buffeli to infect naive calves. These results were consistent with previous vector studies indicating that H.bancrofti and H.humerosa were likely vectors for T.orientalis buffeli in Queensland (Stewart et al., 1987a,b). H. longicornis occurs in the coastal areas of Victoria and New South Wales and extends northwards as far as Gympie in Queensland but is absent from large areas of Northern Australia where Theileria sp (T.orientalis buffeli) is present (Reik, 1982).
Prior inoculation of T.orientalis buffeli-infected blood containing between 6.5 × 106 GC/µL (iv) or 4 × 109 GC/µL (sc) and allowed to “consolidate/incubate” for 13 and 4 weeks, respectively, before T.orientalis ikeda- infected ticks were applied, significantly reduced the initial parasitosis of T.orientalis ikeda over the next 50 days. It was also observed that 2 calves already serendipitously parasitised with < 500 GC/ul T.orientalis ikeda on arrival at 4 months of age, presumably in utero or post-natally, were also significantly “protected” from the first wave of parasitosis following tick challenge. These outcomes consolidated several older theilerial reports from Japan and Korea (Baek et al., 1982; Minami et al., 1981; Onuma et al., 1997) with T.orientalis sergenti, which has been confirmed as T.orientalis (Stewart et al., 1996). A cryopreserved vaccine containing 2 × 108 red blood cells containing T.orientalis [sergenti] per dose “had an inhibitory effect on the clinical manifestation of theileriosis” with a need for proliferation of the inoculum (Ishihara, 1962) but this was not developed further. Production of an attenuated whole blood vaccine against T. orientalis [sergenti] occurred in Korea but outcomes were not reported and challenge appeared to use blood stabilate (Baek et al.,1992). Later, sonicated T. orientalis [sergenti] merozoites produced significant reductions in parasitosis after 3 months, among recipients receiving 2 doses of 100 mg in complete Freund’s adjuvant subcutaneously, 1 month apart, and subjected to field infestation from 2–5 months after initial vaccination (Baek et al., 1992). Unfortunately, the trial was terminated after 5 months as all controls and 20% (4/20) of vaccinates required treatment with diminazene (Berenil®) for anaemia (Baek et al., 1992). A recombinant MPSP vaccine for T.orientalis [sergenti] utilised 3 vaccinations at 3 week intervals, producing an antibody response but no protection against challenge (Park et al, 1999).
Overall, these reports and the current trial support the premise that prior infestation with either T.orientalis ikeda or buffeli merozoites, effectively generates a state of “premunity” which mitigates the severity of a subsequent tick challenge with T.orientalis ikeda (Neal et al., 1969; Stewart et al., 1992). This situation reflects field experience where recovered cattle may harbour multiple theilerial genotypes in the carrier state. It should be noted that inoculation of merozoites of T.orientalis ikeda into susceptible cattle does not appear to cause the clinical episodes of theileriosis that follows tick infestation (Hammer et al., 2016; this study). Such a method of “immunisation” may be a feasible means of control against ticks carrying the virulent theilerial genotypes.
These results are also consistent with the susceptibility of introduced cattle and calves in endemic regions with multiple theilerial genotypes. As noted with the kinetics and age-related infections of calves and introduced stock (Jenkins et al., 2015; Swilks et al., 2017) and in naïve animals at Dorrigo, T.orientalis buffeli infestations are the slowest to develop (Emery et al., unpublished). Results indicated that infections with the virulent genotypes (ikeda and chitose) occurred earlier with maximal parasitosis around 5–6 weeks before declining, whereas parasitosis with T. orientalis buffeli was detectable in an increasing proportion of naïve animals up to 3–4 months before stabilising (Jenkins et al., 2015; Emery et al., unpublished). All animals remained carriers of all four genotypes in the AusDiagnostics® PCR kit for at least 6 months and > 80% of ticks sampled from Dorrigo and Stroud contained DNA from the four theilerial genotypes (ikeda, chitose, Type 5 and buffeli). The earlier appearance of the virulent genotypes negates any opportunity for endemic T.orientalis buffeli to generate any host-protective capability before challenge with virulent genotypes.
This study is the first confirmation of speculations that the carrier state persisting in cattle which have recovered from the initial parasitosis with virulent theilerial genotypes, establishes some type of “protection” to subsequent, seasonal tick challenge. As recommended by de Vos (2011), dose-response studies with single or mixed genotypes are required to establish vaccination potential. Field trials in endemic regions with high levels of tick infestation harbouring multiple theilerial genotypes are also vital to determine the robustness of the method, and whether there is any further “protection” conferred to parasitosis induced by subsequent tick challenges. Therefore, this outcome is being repeated in a field trial providing an intense natural challenge and to test homologous protection using T.orientalis ikeda blood. It may be that prior immunisation with merozoites from any genotype(s) of T.orientalis may reduce the severity of parasitosis following challenge with virulent genotypes as appears to be the case in numerous regions of infested with T.orientalis.
In an additional observation on the vector competence of H.longicornis for T.orientalis ikeda, unfed nymphs infected as larvae effectively transmitted the parasite to naïve second host calves and after collection and moulting to adult stages, could readily transmit T.orientalis ikeda to a further group of naïve calves. This indicated that the tick retained an initial infection acquired in the larval stage, through two moults to the adult stage and did not require any “boosting” from the second host. In the field, this implies that the nymphal stage could engorge on companion or wildlife second hosts and still remain infective for cattle as their third host.