Merozoites of Theileria Orientalis Buffeli Reduce Parasitosis Following Challenge by Ticks Infested with T.Orientalis Ikeda

Background: An investigation aimed to conrm whether immunisation with the “benign” buffeli genotype of Theileria orientalis could reduce the parasitosis of the virulent ikeda genotype. Methods: Calves were inoculated intravenously or subcutaneously with bovine blood containing merozoites of T.orientalis buffeli. When recipients became positive, they and control animals were challenged with unfed nymphs of Haemaphysalis longicornis ticks infested as larvae with T.orientalis ikeda. Results: All calves became positive for the challenge within 12 days after tick application. In the immunised calves, the rst wave of parasitosis with T.orientalis ikeda from 4-6 weeks was reduced signicantly by >80% before the infestation declined into the carrier state by 9 weeks. Conclusions: The results conrm the eld experience with theileriosis in endemic zones where the carrier state appears to prevent clinical disease despite repeated, seasonal tick infestations with virulent genotypes of the parasite. This method offers a means to reduce the severity of the rst wave of theilerial parasitosis after tick challenge and recover associated production losses.


Background
The intracellular protozoal parasite Theileria orientalis has rapidly spread across South-eastern Australia, substantially impacting local cattle industries since 2006. Given that Theileria buffeli, causing "benign theileriosis" had been present in Queensland since 1912 (Stewart et al.,1992;1996), the new syndrome was termed "Bovine Anaemia caused by the T heileria o rientalis group" (BATOG; Eamens et al., 2013a). Molecular techniques have since enabled discrimination of the T.orientalis genotypes and identi ed genotypes "ikeda" and "chitose" as those causing clinical disease (Kamau et al., 2011;Eamens et al., 2013a,b;Bogema et al., 2015). The basis for the current classi cation uses the major piroplasm surface protein (MPSP), which is expressed in the intraerythrocytic stage of T. orientalis and conserved to some extent among different geographic isolates. It has been widely used for molecular epidemiological studies of T. orientalis in Japan (Ota et al., Sivakumar et al., 2012), Korea (Park et al., 2017), Kenya, and Australia (Izzo et al., 2010;Kamau et al., 2011;Bogema et al., 2015). Currently, 11 genotypes of T. orientalis (type 1 or chitose, type 2 or ikeda, type 3 or buffeli, types 4-8, and N1-N3) had been identi ed based on MPSP gene sequences (Sivakumar et al., 2014;Bogema et al., 2015). Of these genotypes, 1 and 2 cause the majority of clinical disease in cattle, with the phylogenetic cluster chitose A mostly associated with the ikeda genotype in clinical cases . On farms impacted by the parasite, NSW-DPI have estimated an average cost of $59K for dairy producers and $11.6K for beef producers, which equates to AUD $131/head for dairy cattle and AUD $67/head for beef cattle (Bailey 2012  Interestingly, inoculation of naïve cattle with blood stages of T.orientalis genotypes does not cause clinical disease (Hammer et al., 2016;Gibson, 2017), but a carrier state is established and parasitosis is detectable by PCR in blood for greater than 30 months (Hammer et al., 2016;unpublished). Irrespective of the theilerial genotype(s) present, the carrier state arising after natural tick-borne infection appears to prevent recurrence of clinical disease following seasonal tick challenge in endemic regions (Izzo etal, 2012;Kamau et al., 2011;Eamens et al., 2013b). This has been witnessed by the progression of the epidemic curve through eastern and southern Australia and New Zealand, with mainly newborn calves and introduced cattle remaining susceptible to clinical disease in endemic zones (Bailey, 2012;. Similarly, the widespread presence of T.orientalis buffeli carrier cattle in Queensland has been attributed to the low prevalence of virulent genotypes and/or clinical theileriosis in that state ( . In endemic regions, infestations of T.orientalis buffeli in susceptible cattle were clearly outpaced by virulent theilerial genotypes ikeda and chitose in both Gloucester and Dorrigo DE, unpublished), negating any opportunity to induce protection. Leaving vector competence aside, some type and level of immunity exists in carrier cattle which resembles "premunity" (Neal et al., 1969), interfering with the severity of subsequent challenge infections. This is witnessed in the long histories of early "vaccinations" against leishmania, malaria, East Coast fever, babesiosis and poultry coccidiosis ("precocious strains") (see McAllister, 2014).
Consistent with the circumstantial and historical evidence for the operation of "premunity" against repeated seasonal tick infestation with T.orientalis, this study examined whether immunisation with blood stages (merozoites) of T. orientalis buffeli could reduce the severity of subsequent T.orientalis ikeda infestation. This study also enabled an opportunity to determine whether H. longicornis nymphal ticks infected as larvae on an infected calf could transmit that infection as nymphs, to an uninfected second host, and then retain that ability to re-infect a third host as adult ticks.

Theilerial stabilates.
T.orientalis ikeda. Blood was collected from the jugular vein of a donor cow into lithium heparin blood collection tubes at Camden NSW, 28 ml of which was subsequently inoculated into a splenectomised calf at the Tick Fever Centre, Wacol, Qld. Approximately 1 month after inoculation, a stabilate (designated J36) was produced using 100 ml of blood collected into sodium heparin mixed with an equal volume of 20% polyvinylpyrrolidone (PVP 40,000; pH 7.2) cryoprotectant solution. This blood mixture was decanted into each of 5 ml cryotubes The cryotubes were then placed in the vapour phase of liquid nitrogen for 15 minutes before the stabilate was lowered into the liquid nitrogen for storage. The stabilate contained 2 × 10 8 merozoites per ml. For infection of ticks, unfed adult H. longicornis ticks which had previously fed on a splenectomised calf that had been inoculated with a thawed 5 ml cryotube of J36 stabilate, were placed on a subsequent splenectomised calf (3604) and transmitted T. orientalis ikeda. Additional clean larval ticks were then fed on this T. orientalis Ikeda-infested calf to themselves become infected. The engorged larval ticks were then collected and allowed to moult to nymphs in the incubator before allpication.
T.orientalis buffeli. A blood stabilate (J46) was similarly prepared from a cow at Bairnsdale, Victoria and stored in liquid nitrogen at the TFC. To prepare the blood inoculum for this trial, splenectomised calf 3584 was inoculated with a thawed 5 ml cryotube of stabilate intravenously and blood collected some months later.

Calves and infection.
The trials in this study conformed with requirements for animal health and well-being under the University of Sydney Animal Ethics Committee (AEC) permit 2018/1328. Fifteen Holstein (neutered male) calves aged 3-4 months were purchased from Leppington Pastoral Company (LPC, Cobbity, NSW) and housed on pasture at The University of Sydney's Pye Farm, Greendale NSW. Calves were weighed and bled to con rm their negative theilerial status by AusDiagnostics® Multiplex-Tandem PCR (MT-PCR) before being randomly assigned to 3 treatment groups, each of 5 animals. Group 1 calves were premedicated with 100 mg of the antihistamine chlorpheniramine maleate (Histamil ®) and 15 mg dexamethasone ( Dexapent®) by intramuscular injection, to lower the risk of any reaction from blood incompatibility. After 30 min, each calf was given a 5 ml intravenous (IV) infusion of fresh infected blood obtained from an infected steer at the TTFC, (steer 3584, infected with T.orientalis buffeli blood stabilate). Previous screening by PCR indicated a speci c infection containing 1.12 × 10 5 gene copies of T.orientalis buffeli per ul blood. The inoculum contained 1.3 × 10 6 merozoites of T.orientalis buffeli per ml. All calves became PCR positive within 28d. Group 2 calves were to be infested with 100 H.bancrofti nymphs (infected with T.orientalis buffeli as larvae) from TFC under patches, but these ticks died on steer 3584. Consequently, H.longicornis larvae were fed on steer 3584, moulted, and approx. 200 unfed nymphs were applied to each calf in Group 2 under backline calico patches as described by Marendy et al. (2019). Ticks were collected and removed after 6 days when they were engorged. When these 5 calves failed to become PCR positive after 5 weeks, 5 ml of fresh infected blood containing 9 × 10 8 T.orientalis buffeli merozoites per mL (from steer 3584) was inoculated subcutaneously (SC) into each calf. All these 5 calves were PCR positive for T.orientalis buffeli within 21d.
Thirteen weeks after Group 1 calves and 28 days after Group 2 calves were inoculated with T.orientalis buffeli blood, approximately 200 unfed H.longicornis nymphs, previously infected with T.orientalis ikeda as larvae at TFC, were placed under backline patches on each of the 15 calves (including the uninoculated group 3 controls). The ticks were collected from each calf after 6 days, counted and pooled, before posting to the Biosecurity Sciences Laboratory (BSL, Brisbane, Qld), to moult to adults; 10 were also placed into 100% ethanol for PCR. When moulted and their exoskeletons had hardened (Marendy et al.,2019), around 50 of these unfed adult H.longicornis were applied to each of 3 naïve calves to determine if the original T.orientalis ikeda infection persisted through the nymphal stage on the uninfected calves and survived through the moult to adult ticks.
All calves were monitored visually daily, with 5 ml blood collected weekly into EDTA vacuum tubes (Vacuette, Griener Bio-one) from day 14 post-inoculation of buffeli blood and from days 14-62 after T. orientalis ikeda infected, tick challenge.

Sample analyses.
PCV was measured by centrifuging blood in micro-haematocrit tubes (Beckman Coulter centrifuge).
Diagnostic PCR was performed by DNA extraction from 200 ul blood samples using the MagMax™ CORE Nucleic Acid Puri cation kit (ThermoFisher Scienti c Inc.). A KingFisher Duo™ Prime Magnetic Particle Processor completed DNA isolation.
Theilerial PCR runs were conducted with 11 eluted DNA samples, one indicator sample, MasterMixes (Theileria), oil, water, and step 2 strips loaded separately into the AusDiagnostics ® Easy-Plex ™ Processor, which provided sequences for theilerial genotypes ikeda, buffeli, chitose, and type 5. Both automatic assay set-up and Step 1 PCR were accomplished in this machine. Samples were then transferred into the Bio-rad CFX96 analyser to perform the Step 2 PCR. Real-time PCR Ct-values were collected for data analysis. Relative gene copy numbers per ul of blood (GP/ul) were calculated for both pan-theileria and theilerial genotypes from spiked standards in the assay.

Statistical analysis.
Statistical analysis was performed using GenStat ™. Raw data were log e transformed and two linear mixed models for PCV and ikeda gene copies per µL were developed for each study as appropriate. The xed effects were Day, Treatment and the interaction between Day and Treatment. The random effect was Animal ID. P-values were calculated for each xed effect in each model to determine signi cance.
For signi cant xed effects, the differences in the predicted means for each factor level were compared to the Least Signi cant Differences (LSDs) at signi cance level 0.05 to determine whether pairwise comparisons were signi cant. If the interaction xed effect was signi cant, no further pairwise comparisons were determined for the other xed effects.
Predicted means for PCV and GC/ul for theilerial genotypes within treatment groups were presented with standard error (SE) bars.

Immunisation with T.orientalis buffeli.
Both intravenous (IV) and subcutaneous (SC) inoculation of bovine blood infected with T.orientalis buffeli produced parasitosis detectable by PCR within 4 weeks of inoculation. Over the 13 weeks before challenge, the parasitosis in Group 1 (IV) reached a mean peak of 5,063 GC/ul of T.orientalis buffeli by 5 weeks, decreasing and stabilising 9-13 weeks after initial inoculation between means of 1292 and 876 GC/ul (Table 1). By comparison, at the time of challenge, 4 weeks after inoculation SC, Group 2 calves had a mean parasitosis of 1547 GC/ul (Table 1). Following the T. orientalis Ikeda infected, tick challenge, the parasitosis with T.orientalis buffeli remained relatively steady in both Groups 1 and 2 over the next 60 days at < 2500 GC/ul. Results are expressed as mean GC/ul blood +/-SE.

* 4 weeks after inoculation of blood IV; na = not applicable
Following the initial inoculations of blood into Group 1, it was revealed that 2 calves in the control Group 3 exhibited pre-existing low parasitoses with T.orientalis ikeda of < 500 GC/ul, presumably from infections acquired in utero or postnatally. This meant that these 2 calves were excluded from the control group (3 calves) for statistical analysis, but were included for analysis as a separate group (of 2 calves) after tick infestation.

Effects of T.orientalis buffeli on T.orientalis ikeda after tick challenge.
All 15 calves in the 3 groups became positive for T.orientalis ikeda within 12 days after infestation (DAI) from the application of the 200 infected H.longicornis nymphs. The parasitosis in the control groups followed a typical pattern in peaking around 5 weeks (39 DAI) after infestation at a mean 69734 GC/ul before declining to < 2000 GC/ul blood by 62 DAI (Fig. 1, Table 2). The parasitosis in the 3 treatment groups were signi cantly reduced between 30 and 85% on sampled days during the rst wave of parasitaemia from 25-39 DAI (Fig. 1, Table 2). In parallel, the PCV in the control group decreased by 16-20% after infestation to a mean of 25% by 39 DAI, signi cantly reduced compared to the SC immunised group 2 (Table 3) and remained signi cantly lower than group 2 up to 62 DAI ( Table 3).The PCV of the IVimmunised group 4 was only signi cantly higher than control group 1 on 32DAI (Table 3).

Discussion
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 × 10 6 GC/µL (iv) or 4 × 10 9 GC/µL (sc) and allowed to "consolidate/incubate" for 13 and 4 weeks, respectively, before T.orientalis ikeda-infected ticks were applied, signi cantly 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 signi cantly "protected" from the rst 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 con rmed as T.orientalis (Stewart et al., 1996). A cryopreserved vaccine containing 2 × 10 8 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 signi cant 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 eld 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 re ects eld 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 ( This study is the rst con rmation 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 eld 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 eld, this implies that the nymphal stage could engorge on companion or wildlife second hosts and still remain infective for cattle as their third host.

Conclusions
Calves inoculated SC or IV with blood infested with merozoites of T.orientalis buffeli became positive for the parasite by PCR around 28 days after infestation (DAI) and did not develop clinical signs over the following 9 weeks. Following the application of 200 H.longicornis nymphs harbouring T.orientalis ikeda, the rst peak of parasitosis occurring from 25-47 DAI was reduced by around 80% in immunised calves compared to control animals. These results are consistent with the lack of clinical theileriosis in carrier cattle in endemic regions and strongly suggest that host regulation maintains theilerial populations of the virulent genotypes below levels causing clinical disease, despite repeated seasonal tick challenges. The results also imply that prior vaccination with blood forms of the parasite could reduce deaths and production losses incurred from the rst wave of parasitosis following tick infestation with virulent theilerial genotypes ikeda and chitose A.

Declarations
Competing interests. The authors declare that they have no competing interests