Dynamics and within-host interaction of theileria lestoquardi and t. Ovis among naive sheep in oman

ABSTRACT Mixed species infections of _Theileria _spp. are common in nature. Experimental and epidemiological data suggest that mixed species infections elicit cross-immunity that can

modulate pathogenicity and disease burden at the population level. The present study examined within-host interactions, over a period of 13 months during natural infections with two

_Theileria _spp., pathogenic (_T. lestoquardi_) and non-pathogenic (_T. ovis_), amongst a cohort of naive sheep in Oman. In the first two months after exposure to infection, a high rate of

mortality was seen among sheep infected with _T. lestoquardi_ alone. However, subsequently mixed-infections of _T. lestoquardi_ and _T. ovis_ prevailed, and no further death occurred. The

overall densities of both parasite species were significantly higher as single infection vs mixed infection and the higher relative density of pathogenic _T. lestoquardi_ indicated a

competitive advantage over _T._ ovis in mixed infection. The density of both _species_ fluctuated significantly over time, with no difference in density between the very hot (May to August)

and warm season (September to April). A high degree of genotype multiplicity was seen among _T. lestoquardi_ infections, which increased with rising parasite density. Our results illustrate

a potential competitive interaction between the two ovine _Theileria _spp., and a substantial reduction in the risk of mortality in mixed parasite infections, indicating that _T. ovis_

confers heterologous protection against lethal _T. lestoquardi_ infection. SIMILAR CONTENT BEING VIEWED BY OTHERS INFECTION WITH _BORRELIA AFZELII_ AND MANIPULATION OF THE EGG SURFACE

MICROBIOTA HAVE NO EFFECT ON THE FITNESS OF IMMATURE _IXODES RICINUS_ TICKS Article Open access 21 May 2021 ASYMPTOMATIC VIRAL INFECTION IS ASSOCIATED WITH LOWER HOST REPRODUCTIVE OUTPUT IN

WILD MINK POPULATIONS Article Open access 09 June 2023 INVESTIGATION OF VERTICAL AND HORIZONTAL TRANSMISSION OF _SPIROPLASMA_ IN TICKS UNDER LABORATORY CONDITIONS Article Open access 15

August 2023 INTRODUCTION Malignant Ovine Theileriosis (MOT), caused by _Theileria lestoquardi,_ in sheep and Tropical Theileriosis, caused by _Theileria annulata_ in cattle, are widespread,

tick-borne diseases in tropical and subtropical regions. As for other apicomplexan parasites, mixed infections of different _Theileria _spp. and genotypes of the same species is

frequent1,2,3,4,5,6,7, due to sequential infection in areas of high transmission intensity or simultaneous infection of multiple genotypes from a single tick. The acquisition of multiple

parasites is often associated with interactions that can influence the outcome of disease and the fate of each parasite within the infected host. Within-host competitive interaction between

parasites is a major evolutionary force that can shape the parasite populations and disease outcome. It may affect parasite density, transmission and virulence8. Understanding how the

co-infecting parasites interact is central to understanding transmission dynamics and disease risk at the population level. A plausible mechanism postulated to drive parasite-parasite

interaction is modulation of the host’s immune system that results in an enhanced response against other pathogens8,9. The influence of co-infection of one parasite on the infection outcome

of another has been called “heterologous reactivity”, i.e. immunity to one pathogen reducing susceptibility to a second9. Consequently, the density and disease outcomes of virulent parasite

strains may be less in mixed infections than in a single infections10,11. Studies in rodent malaria parasites have demonstrated that, in immune competent mice the avirulent _Plasmodium_

strain suffered more competition than a more virulent strain, demonstrating a competitive advantage of virulent parasites in an immune-mediated interaction11. Similarly, an experimental

study on _T. annulata_, showed higher density of the virulent clone in a mixed compared to a single infection12. This study reports the first detailed longitudinal investigation of the

dynamics (within-host interactions) of coinfection of two _Theileria species_, the pathogenic parasite, _T. lestoquardi_, the causative agent of MOT and the non-pathogenic, _T. ovis_, in a

naïve cohort of sheep in Oman. Our previous studies in Oman revealed a high prevalence of _Theileria _spp. among livestock13. MOT in sheep is associated with high morbidity of 30–40% and

mortality can reach up to 100% among clinical cases of indigenous breeds during seasonal epidemics14. However, a large proportion of sheep carry asymptomatic mixed species infections of _T.

lestoquardi_ and _T. ovis_7. The data suggest the occurrence of interactions between the pathogenic and less pathogenic species of _Theileria_ and supports the hypothesis that concurrent

co-infection can lead to a reduction in parasitaemia and mortality associated with the pathogenic species9. This premise was tested further in the current study. RESULTS EFFICIENCY AND

SENSITIVITY OF QPCR FOR QUANTIFICATION OF _T. LESTOQUARDI_ AND _T. OVIS_ _T. lestoquardi_ and _T. ovis_-specific qPCR assays were developed to estimate parasite density and investigate

within-host interaction between the two ovine _Theileria _spp. endemic in Oman. Initially, assay indices considered indicative of a well-optimized qPCR were assessed. This included the

linearity of data (_R_2 > 0.98), an efficiency (E) value within the range of 80–100% and consistency of Cq values across replicates. The qPCR amplification efficiency was 91% and 82% for

_T. lestoquardi and T. ovis 18s rRNA_ genes, respectively. The inter-assay variability between standard curves was 2% and 1% for the _T. lestoquardi 18s rRNA_ and _T. ovis 18s rRNA_ assays,

respectively, while the correlation between log10 _18s rRNA_ copies and Cq values was significant for both species (_T. lestoquardi_ adjusted R2 > 0.99 for all PCRs with _P_ < 0.001;

_T. ovis_ adjusted R2 > 0.98 for all PCRs with _P_ < 0.001). A high consistency of Cq values across replicates was seen, the standard deviation15 ranged between 0.001–0.3 and

0.0003–0.3 for _T. lestoquardi_ and _T. ovis 18s rRNA_ qPCR, respectively. The limit of detection3 was 9.26 log10 and 5.3 log10 _T. lestoquardi_ and _T. ovis 18s rRNA_ copies/μl blood,

respectively (Fig. 1). ANALYSIS OF _T. LESTOQUARDI_ AND _T. OVIS_ INFECTED SHEEP Fifty sheep (North Oman breed) were transferred to an endemic farm in April 2016. There was minimal

difference in the mean age of the examined sheep (31–32 weeks) and all sheep were kept exclusively indoors, in a small open enclosure attached to the farmer’s residence, with similar

expected levels of exposure to ticks, _Hyalomma anatolicum_. At the early stages of the study, May and June 2016, 3 and 4 sheep died of suspected MOT, respectively. _Theileria_ species

identification and density were determined in blood samples collected from 43 sheep between May 2016 and May 2017 (13 times points per sheep). No parasites were seen when examined by Giemsa

stained blood film and microscopy for the majority of the samples. However, occasionally animals showed parasites detected by microscopy during the follow up period. Interestingly, a very

high parasite density (piroplasm stage) was seen in one dead animal examined few hours prior to death. Therefore, _Theileria ssp_ were detected and infection levels assessed primarily by

qPCR. Infection was defined as single species, when the initial infection in May 2016 was diagnosed with either _T. lestoquardi_ or _T. ovis_ alone and continued in consecutive months till a

mixed species infection (_T. lestoquardi_ plus _T. ovis_) was seen. As the outcome of interaction between the two species can be influenced by the immune response9, the appearance of a

single species following the detection of mixed species was not defined as single infection. _Theileria _spp. could be detected in 485 (91%) of the 533 (95%) blood samples, collected during

the study period; _T. lestoquardi_ alone was detected in 125 (23%) and _T. ovis_ alone was detected in 4 (1%) of the samples. Thus, a mixture of both species was detected in the majority of

blood samples. The lower prevalence of single _T. ovis_ differs from an earlier survey in Oman, (April and August 2014) when single _T. ovis_ infection was more dominant than single _T.

lestoquardi_6. The lower prevalence of single _T. ovis_ among the present cohort is unlikely to be due to technical deficiency generating false negative results, as the limit of detection of

the _T. ovis_ specific qPCR assay was two-fold higher than that of _T. lestoquardi_. PATTERN OF THEILERIA SPECIES INFECTION AND MORTALITY Figure 2 shows the pattern of _T. lestoquardi_ and

_T. ovis_ infection and mortality in a cohort of sheep (n = 50), over a period of 13 months (May 2016 to May 2017). At the start of the study (April 2016) 50 apparently healthy and

uninfected sheep were transferred to a known area of _Theileria_ transmission. In May 2016 (second sampling point) all animals had become infected; 62% had single _T. lestoquardi_ infection,

38% had mixed infection (_T. lestoquardi_ plus _T. ovis_), and no single _T. ovis_ infections were detected. At this point, 3 sheep died, and an additional 4 animals died in June 2016. In

subsequent sampling of the remaining 43 animals, single _T. lestoquardi_ infections decreased dramatically reaching 2% by March 2017 and zero in May 2017. In contrast, mixed infections

increased from 38% in May 2016 to 100% in May 2017 (Fig. 2, Supplementary Table 1). Disease symptoms (enlarged superficial lymph nodes, high fever and anorexia) consistent with theileriosis

were observed in the first two months (May and June 2016). During this period, there were cases of mortality (7 out of 50 animals) and a high rate of single _T. lestoquardi_ infection (50.8%

of cohort). No other pathogens or clinical signs associated with ovine diseases known to occur in the study region were recorded. Parasite infection was detected by PCR and examination of

the 7 dead showed _T. lestoquardi_ schizonts and piroplasms in a few animals. Investigation of the 7 dead animals revealed enlarged superficial lymph nodes, while necropsy of one animal also

showed an enlarged liver and spleen. Potential increase in overt clinical signs assessed in surviving single infected animals may have occurred earlier in the infection period, prior to

death, between the two time points, but were not taken. All but one of the animals that died, were infected by _T. lestoquardi_ alone, and mortality was significantly associated with single

_T. lestoquardi_ infection (Fisher’s exact test, _P_ < 0.001). The parasite population seen in each of the dead animals, prior to death, was comprised of a different, distinct combination

of multiple genotypes, as identified by the pattern of alleles generated by five neutral microsatellite markers (Fig. 2, Supplementary Table 1). Thus a specific virulent population or over

representation of certain genotypes could not be associated with death in these animals. DYNAMICS AND INTERACTION BETWEEN _T. LESTOQUARDI_ AND _T. OVIS_: PARASITE INFECTION Following the

acute phase of MOT, 43 (86%) sheep maintained chronic asymptomatic _Theileria _spp. infection throughout the 13 months of the study period, most often as a mixed infection detectable by

qPCR. Figure 3 shows the persistence and temporal dynamics of _T. lestoquardi_ and _T. ovis_ detected among sheep with mixed species infection, using _18s rRNA gene_ copy number estimated by

qPCR as a proxy measurement of parasite density. The estimated density of _T. lestoquardi_ fluctuated significantly during the study period (log likelihood: − 636.91, df: 10, _x__2_:

51.582, _P_ < 0.001), ranging between 2.8 log10 _18s rRNA_ copies/μl (95% CI 1.3–4.3 log10) in May 2016 to 5.1 log10 _18s rRNA_ copies /μl blood (95% CI 4.9–5.4 log10) in May 2017. A

similar pattern was seen for _T. ovis_ (Log likelihood: − 571.9, df: 10 _x__2_: 39.77, _P_ < 0.001) (Fig. 3). When the study period was divided between the two distinct climatic seasons

of the region; very hot (May to August) and warm (Sept to April), no seasonal linked changes in density were seen for either parasite; _T. lestoquardi_ (log likelihood: − 661.58, df: 1,

_x__2_: 2.254, _P_ = 0.133) and _T. ovis_ (log likelihood: − 591.83, df: 1, _x__2_: 0.086, _P_ = 0.769). The estimated average _T. lestoquardi_ density was slightly higher (4.7 log10 _18s

rRNA_ copies/μl blood, 95% CI 4.5–4.9 log10) in the warm season compared to the very hot season (4.5 log10 _18s rRNA_ copies/μl blood, 95% CI 4.0–4.9 log10). Conversely, for _T. ovis_ the

estimated average density was slightly lower (3.7 log10 _18s rRNA_ copies/μl blood, CI 3.4–4.1 log10) in the warm season compared to the hot season (4.5 log10 _18s rRNA_ copies/μl blood, CI

4.1–4.9 log10). INTERACTION BETWEEN _T. LESTOQUARDI_ AND _T. OVIS_ IN MIXED INFECTION The detection of mixed infections of _T. lestoquardi and T. ovis_ increased from 36% in May 2016 to over

80% by Sep 2016 among the examined sheep and all animals with mixed infection, as well as the few persisting single _T. lestoquardi_ infections, remained asymptomatic and apparently healthy

(Fig. 2). We therefore tested whether the interaction between _T. lestoquardi_ and _T. ovis_ among these animals was associated with modulation of the load of either parasite. The estimated

_T. lestoquardi_ density was higher (Log likelihood: − 662.7, df: 1, _x__2_: 0.014, _P_: 0.90) in single infection (5.2 log10 _18s rRNA_ copies/μl blood, 95% CI 4.9–5.5 log10), compared to

mixed infection (4.5 log10 _18s rRNA_ copies/μl blood, 95% CI 4.2–4.8 log10). In addition, differences in _T. lestoquardi_ densities in single vs mixed infection was more pronounced,

generally, over time, when time as a variable was added to the model (Log likelihood: − 630.08, df: 9, _x__2_: 13.658, _P_ < 0.001). For example, the mean _T. lestoquardi_ density in

single infection in May 2016 and in March 2017 (4.4 and 5.9 log10 _18s rRNA_ copies /μl blood, 95% CI: 3.6–5.2 log10) were clearly higher than the density of mixed infections (0.4 and 5.1

log10 _18s rRNA_ copies /μl blood, 95% CI: − 0.5–1.4 log10) (Fig. 4). Where detected (4 time points), single infection _T. ovis_ density was significantly greater compared to mixed infection

with _T._ _lestoqurdi_ for the same time points (Log likelihood: − 426.44, dF: 10, _x__2_: 291.07, _P_ < 0.001). However, in mixed infections, _T. lestoquardi_ was detected at higher

density than _T. ovis_ in the majority of the examined time points (Fig. 3). MULTIPLICITY OF _T. LESTOQUARDI_ GENOTYPES Previous analyses generated evidence of a high rate of multiplicity of

_T. lestoquardi_ genotypes in infected sheep in Oman7. The multiplicity of infection detected by five polymorphic microsatellites among animals in the current study ranged between 80.8% and

100%. In the first month of the study, 38 (80.8%) out of the 47 sheep examined were infected with multiple genotypes of _T. lestoquardi_. At this time, the estimated minimum mean number of

genotypes per sheep among the cohort was 2.1. Genotype multiplicity and therefore the mean number of clones/genotypes (MNC) of _T. lestoquardi_ per infected sheep increased significantly

over time (Log likelihood: − 460.88, df: 10, _x__2_: 48.538, _P_ < 0.001), from 2.1 (95% CI 1.8–2.4) in May 2016 to reach 2.9 (2.6–3.1) and 3.0 (95% CI 2.7–3.2) in September 2016 and

March 2017, respectively. This suggests the occurrence of superinfection over time via successive infestations of multiple infected ticks. _T. lestoquardi_ density positively associated with

MNC (Log likelihood = − 615.46, df: 1, χ2: 20.55, _P_ < 0.001): _T. lestoquardi_ specific 18rRNA copies/μl DNA, was 3.54 (95% CI 2.60–4.48), 4.49 (95% CI 4.26–4.72), 4.86 (95% CI

4.70–5.01), 4.93 (95% 4.70–5.16) and 5.21 (95% CI 4.43–5.99), when the MNC was 1, 2, 3, 4 and 5 clones/genotypes respectively (Fig. 5). There was no difference in MNC between single _T.

lestoquardi_ 2.64 (95% CI 2.45–2.84) and mixed infections (_T. lestoquardi _and _T. ovis_) 2.64 (95% CI 2.55–2.74) (Log likelihood = − 463.42, df: 1, χ2: 1.048, _P_ = 0.306). This suggests

similar levels of diversity of _T. lestoquardi_ in animals with single and mixed species infection. DISCUSSION This natural challenge study revealed the long-lasting persistence (over a

period of 13 months) of _T. lestoquardi_ and _T. ovis_ parasites as asymptomatic mixed species infections in naïve sheep in Oman, and provided evidence of competitive interactions between

these two parasites. The more pathogenic _T. lestoquardi_ was associated with higher density and mortality when present as a single infection relative to mixed infection with the

non-pathogenic _T. ovis_. Thus, co-infection with the two parasites species correlated with reduced mortality of MOT in sheep_,_ supporting the hypothesis of heterologous protection proposed

for other combinations of _Theileria_ spp. mixed infection 9. _T. lestoquardi_ was associated with higher density when present as a single infection than where it was mixed with the

non-pathogenic _T. ovis_ (Figs. 3 and 4). A similar pattern was seen with _T. ovis_ in mixed infection compared to the few occasions when it existed as single infection (4 time points),

demonstrating within host interaction between the two parasites. This is evident by the fact that density of both species in mixed infection fluctuated around a threshold over the study

period, while the animals were apparently healthy, with a tendency to return to an equilibrium value (Fig. 3), indicating a cross-species mechanism of regulation_._ In mixed parasite

infection, within-host interactions resulting in lower virulence can occur through several mechanisms, including heterologous innate immunity9. Within-host selection may also favour traits

that allow the parasite to avoid immune responses and survive at low densities16. A low degree of serological cross-immune reactivity has been detected between _T. ovis_ and _T. lestoquardi_

together with the relatively long duration of a detectable antibody response17, but it is unclear whether this provides cross protection against disease. There is evidence that immunity to

_T. lestoquardi_ in sheep involves T cell mediated responses18 against the macroschizont infected leukocyte and similar immune responses have been reported for _T. annulata_ and _T.

parva_19. Genetic and antigenic similarities are evident for _T. lestoquardi_ and _T_. _annulata_, and cross-immunity between the two species has been demonstrated17,20. However the disease

produced by these parasites are distinct from _T. ovis_, where amplification of the macroschizont infected leukocyte does not occur to any known extent, and replication is more apparent

within erythrocytes similar to _T. orientalis_21. Thus, it is debatable whether the reduced parasite density in mixed infections found in this study relate to an acquired immune response:

non-specific mechanisms linked to innate immunity and/or growth inhibition should be considered for further investigation. However, _T. lestoquardi_ was associated with higher density when

present as a mixed infection with the non-pathogenic _T. ovis_. This suggests a prevailing competitive advantage of _T. lestoquardi_ over the non-pathogenic, _T. ovis_. This is evident by

the relatively lower density of _T. lestoquardi_ in mixed infection compared to single infection. The relative density pattern in mixed species infection could be, explained by a competitive

advantage of the pathogenic parasite (_T. lestoquardi_) in an immune-selection (potentially via innate immunity) environment, where it is more likely to overcome nonspecific immune

responses, and build up higher densities22. However, the suppressive environment of mixed infection on _T. ovis_, has not been tested, as few animals in cohost displayed single _T. ovis_.

Nonetheless, on the few occasions when single _T. ovis_ infection (n = 4) appeared during the follow up period, as in these few samples _T. ovis_ density was always relatively higher

compared to mixed infection (Samples, 3981, 3986, 3988 and 3995; Supplementary Table 1). The above findings may be consistent with experimental studies using genetically-distinct strains of

the rodent malaria parasite, _Plasmodium chabaudi,_ in mice, where the more virulent parasite competitively suppresses a less virulent one in mixed infection11. Similarly, observational data

from multiple _Plasmodium_ parasites also suggest the existence of competitive suppression23. The dynamics of multispecies _Plasmodium_ infection in asymptomatic carriers, under intense

transmission provide evidence for a density-dependent regulation that transcends species as well as genotypes23. Whether this form of interspecies competition operates in ovine Theileria

infection, or one species is simply more resistant to detrimental conditions than the other requires further study. The delayed appearance of _T. ovis_ among the examined cohort (Fig. 3) was

not expected from previous reports on species prevalence, but may be attributed to differences in the kinetics of proliferation of the two parasites. _T. ovis_ parasites are not detected in

the blood until the large macroschizont stages mature and merozoites invade erythrocytes, whereas for _T. lestoquardi,_ asexual multiplication occurs within leukocytes within the first week

of infection with parasites entering red blood cells in week two24. Nevertheless, despite the possibility that _T. lestoquardi_ may competitively suppress _T. ovis_ in mixed infections, _T.

ovis_ is widespread in Oman6, indicative of a high transmission capacity. A high level of _T. ovis_ transmission is also reflected in the increased rate of mixed infection over time seen

our study. The two parasites are transmitted by _Hyalomma anatolicum_, which was the main tick species identified on the examined animals and is widely distributed in Oman6. A high level of

genetic diversity and genotype multiplicity of _T. lestoquardi_ was detected in samples from the infected cohort of sheep. Almost all infected animals displayed a novel (or combination of)

genotype (defined by unique combinations of alleles detected by the examined microsatellites), including strains extracted from dead animals. This is consistent with the high genetic

diversity and multiplicity of infection reported for _T. lestoquardi_ populations in Oman7 and Sudan25. Moreover, the observed increase in _T. lestoquardi_ multiplicity of infection over

time can be attributed to super-infection and the consecutive acquisition of novel genotypes combined with delayed clearance of initial infecting parasites. The study site is known for high

abundance and infestation of _Hyalomma anatolicum_6, and this is predicted to promote cross-mating, genetic recombination and generation of novel parasite genotypes. Multiple genotypes were

common, among a single and mixed _T. lestoquardi_ infection, while total parasitemia exhibited fluctuation around a limit (Fig. 4), suggesting a similar mechanism for regulation of the

dynamics of species- and/or genotypes. The similar level of _T. lestoquardi_ diversity detected in animals with single and mixed species infection suggests that both forms of infection are

equally susceptible to establishment of distinct multi genotype populations. The frequent cross-mating, and random re-assortment of alleles on different gens, generate novel genotypes in

infected ticks which can readily infect sheep. Following the acute phase of infection, however, protective immunity acquired by the animals does not shield from reinfection, a scenario

similar to that reported for _Plasmodium_26. However, over time chronic multi-clonal (mixed genotypes) super-infection is considered to promote a status of ‘premunition’ with elevated

protection against a re-occurrence of disease26, leading to higher genotype complexity in these individuals. Such findings are in agreement with the significant increase in level of _T.

lestoquardi_ genotypic diversity in animals, with both mixed and single infection, without mortality, over time among the examined cohort in this study. In conclusion, the limited data from

this study on the outcome of ovine theileriosis in Oman indicates that single _T. lestoquardi_ infection is associated with high mortality, and that mixed species infections are associated

with _T. lestoquardi_ density regulation, indicative of within-host interaction, and lower mortality. A protective effect of mixed species infection (_T. lestoquardi_ + _T.ovis_) against

severe MOT, are in line with the results of a field epidemiological study in indigenous African calves that demonstrated an 89% reduction in mortality due to _T. parva_ infection (East Coast

Fever) in the presence of less pathogenic _Theileria _spp. (_T. mutans_ and _T. velifera_)9. Further studies of mixed infections of _T. lestoquardi_ and _T. ovis_ are required to validate

whether heterologous protection significantly reduces the impact of morbidity and mortality of MOT. In addition, the role of mixed infection in the epidemiology of ovine theileriosis should

be investigated to determine whether infection of the non-pathogenic _T. ovis_ can be utilised as a predictor, and control strategy, for improved clinical outcome. MATERIAL AND METHODS STUDY

AREA AND SUBJECTS This study was conducted in Sharqiyah province, eastern Oman, where our previous baseline studies identified high _Theileria _spp. (_T. annulata_, _T. lestoquardi_ and _T.

ovis_) infection rates among cattle (T. annulata only, 72%), sheep (_T. lestoquardi_ and _T. ovis_, 50%) and goats (_T. lestoquardi_ and _T. ovis_, 9.1%)6. A cohort of 50 new-born

(3-months-old) local Omani sheep (North Oman breed; 35 males and 15 female) was raised on a tick-free farm at Sultan Qaboos University and then transferred to a farm endemic for theileriosis

in Sharqiyah province, in April 2016. The North Oman sheep breed is indigenous to Oman and is considered more tolerant of _Theileriosis_ than other imported breeds14. Animals acquired

_Theileria_ infection naturally and clinical data and blood samples for DNA extraction were collected every month over a period of one year (May 2016 to May 2017). ETHICAL STATEMENT The

project adhered to the guidelines and ethics code of animal welfare, approved by the Animal Care and Use Committee of Sultan Qaboos University, Oman. The blood samples were collected under

supervision of a veterinarian, a non-invasive method was used to restrain animals, and blood samples collected by experienced technical staff of the Ministry of Agriculture and Fisheries,

Oman. The blood samples collected solely for the purpose of this study and have not been used or will be used for another purpose. The volume of blood collected from each animal, at each

time point, is compliance with the guidance of the Animal Care and Use Committee of Sultan Qaboos University, Oman. Informed consent was taken from the farm owner before drawing blood from

animals. This research was also performed in accordance with the relevant guidelines and regulations of ethical and animal welfare of the Ministry of Agriculture and Fisheries, Oman.

DETECTION AND QUANTIFICATION OF _T. LESTOQUARDI_ AND _T. OVIS_ The density of _T. lestoquardi_ and _T. ovis_ parasites in whole blood was quantified using a real-time polymerase chain

reaction (qPCR) assay. Primers and probes targeting _18S rRNA gene_ of _T. lestoquardi_ and _T. ovis_ were designed (Table 1) using available GenBank sequences for _T. lestoquardi_ and _T.

ovis_, as described6. The specificity of _T. lestoquardi and T. ovis 18S rRNA gene_ primers was tested against pure _T. lestoquardi_, _T. ovis_ and _T. annulata_ DNA samples. The _T.

lestoquardi 18S rRNA_ primer amplified _T. lestoquardi_ DNA samples while no amplification was detected with _T. ovis_ and _T. annulata_ DNA. Similarly, _T. ovis 18S rRNA_ gene primers

amplified _T. ovis_ DNA but not _T. lestoquardi_ or _T. annulata_ DNA. For assay optimization, genomic DNA was extracted from cultured _T. lestoquardi_ and a natural infection with _T. ovis.

The 18S rRNA_ genes were amplified using primers described in Table 1, and purified using Wizard SV Gel and a PCR clean-up system (Promega). Purified PCR product concentration was measured

by Spectrophotometry (Nanodrop), and the copy numbers of _18S rRNA_ genes for both species were calculated using the following equation. DNA concentration (ng) × 6.022 × 1023/ length of DNA

product (bp) × 1 × 109 × 65027. The estimated _18S rRNA_ gene copies were 9.26 × 1010 /μl DNA and 5.3 × 1010 /μl DNA for _T. lestoquardi_ and _T. ovis_, respectively. Eight points of tenfold

serial dilutions (TL: 9.26 × 107–9.26 _18s rRNA_ gene copy/μl DNA: TO: 5.3 × 107–5.3 _18s rRNA_ gene copy/μl DNA) were used to generate standard curves. DNA was extracted from 200 µl blood

of the examined/sampled sheep, using the QIAamp DNA mini kit (Qiagen). Real-time quantitative PCR was performed in Micro Amp Fast optical 96-well reaction plates using the ABI PRISM 7500

Sequence Detection System (Applied Biosystems) with 3 μl of the extracted DNA in 20 μl PCR mix containing 10 μl of 2X TaqMan Universal PCR Master Mix, 0.6 μl of 10 µM of each primer and 0.2 

μl of 10 µM probe. The reaction temperature profile was 50 °C/2 min, 95 °C/10 min, 45 cycles of 95 °C/15 s and 60 °C/1 min. GENOTYPING OF _T. LESTOQUARDI_ Five polymorphic _T. lestoquardi_

specific microsatellites, TL_MS05, TL_MS07, TL_MS13, TL_MS280 and TL_MS281 were used to genotype samples, as described previously7. Multiplicity of infection was then determined as any

infection with more than one allele at the examined loci, and the minimum number of clone/genotypes (MNC) defined by the maximum number of alleles at any locus per infection. STATISTICAL

ANALYSIS Generalized linear mixed models (GLMMs) were used to estimate; (i) changes in prevalence and densities of _T. lestoquardi_ and _T. ovis_ over time4, (ii) temporal change in _T.

lestoquardi_/ _T. ovis_ density among single infections (_T. lestoquardi_ alone or _T. ovis_ alone) and mixed species infections (_T. lestoquardi_ plus _T. ovis_) and (iii) association of

_T. lestoquardi_ MOI and _T. lestoquardi_ density in single and mixed species infections. RESPONSE VARIABLES AND MODEL FITTING STRATEGY A variety of distributions (Binomial, Negative

binomial, Poisson and Gaussian, gaussian zero-inflation) were tested to describe the distribution of _T. lestoquardi_ and _T. ovis_ density between animal hosts. A Gaussian and Gaussian zero

inflation distribution gave the most parsimonious fit for _T. lestoquardi_ count data (Log Likihood: − 662.70, _x__2_:178.220, _P_ < 2e-16) and for _T. ovis_ data (Log Likihood: − 

591.87, x2: 418.4, _P_ < 2.2e−16), respectively. Zero inflation parameters considered the fact that many samples were negative for _T. ovis_ at different time points, especially during

the first months of the experiment. The _T. lestoquardi_ and _T. ovis_ density was allowed to vary between hosts by including them as a random effect in the models. This facilitates

detection of changes in density over time, as there is substantial inter-host variability. Models were built using the forward stepwise selection method and the Log likelihood ratio test

(LRT) was used to determine the most parsimonious fit. When more than one model significantly fitted the data, the one with the lowest Akaike Information Criterion (AIC) was selected28.

EXPLANATORY VARIABLES Fixed effect variables were (i) month of follow-up4, complexity of infection, mixed infection (_T. lestoquardi_ plus _T. ovis_) or single infection (_T. lestoquardi_

alone or _T. ovis_ alone) at any time point during the study and (iii) multiplicity of infection of _T. lestoquardi_ and the minimum number of clones, defined as the maximum number of

alleles across the examined five microsatellites per infection. REFERENCES * Mayxay, M., Pukrittayakamee, S., Newton, P. N. & White, N. J. Mixed-species malaria infections in humans.

_Trends Parasitol._ 20(5), 233–240 (2004). Article  PubMed  Google Scholar  * Iseki, H. _et al._ Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the

simultaneous detection of bovine Babesia parasites. _J. Microbiol. Methods_ 71(3), 281–287 (2007). Article  CAS  PubMed  Google Scholar  * Jalali, S. M., Jolodar, A., Rasooli, A. &

Darabifard, A. Detection of _Theileria lestoquardi_ cross infection in cattle with clinical theileriosis in Iran. _Acta Parasitol._ 61(4), 756–761 (2016). Article  PubMed  Google Scholar  *

Zaeemi, M., Haddadzadeh, H., Khazraiinia, P., Kazemi, B. & Bandehpour, M. Identification of different Theileria species (_Theileria lestoquardi_, _Theileria ovis_, and _Theileria

annulata_) in naturally infected sheep using nested PCR–RFLP. _Parasitol. Res._ 108(4), 837–843 (2011). Article  PubMed  Google Scholar  * Györke, A., Pop, L. & Cozma, V. Prevalence and

distribution of Eimeria species in broiler chicken farms of different capacities. _Parasite_ 20, 1–8 (2013). Article  Google Scholar  * Al-Fahdi, A. _et al._ Molecular surveillance of

Theileria parasites of livestock in Oman. _Ticks Tick-borne Dis._ 8(5), 741–748 (2017). Article  PubMed  Google Scholar  * Al-Hamidhi, S. _et al._ _Theileria lestoquardi_ displays reduced

genetic diversity relative to sympatric _Theileria annulata_ in Oman. _Infect. Genet. Evol._ 43, 297–306 (2016). Article  PubMed  Google Scholar  * Vaumourin, E., Vourch, G., Gasqui, P.

& Vayssier-Taussat, M. The importance of multiparasitism: examining the consequences of co-infections for human and animal health. _Parasites Vectors_ 8(1), 545 (2015). Article  PubMed 

PubMed Central  Google Scholar  * Woolhouse, M. E. _et al._ Co-infections determine patterns of mortality in a population exposed to parasite infection. _Sci. Adv._ 1(2), e1400026 (2015).

Article  ADS  PubMed  PubMed Central  Google Scholar  * Mackinnon, M. J. & Read, A. F. Genetic relationships between parasite virulence and transmission in the rodent malaria Plasmodium

chabaudi. _Evolution_ 53(3), 689–703 (1999). Article  PubMed  Google Scholar  * de Roode, J. C. _et al._ Virulence and competitive ability in genetically diverse malaria infections. _Proc.

Natl. Acad. Sci._ 102(21), 7624–7628 (2005). Article  ADS  PubMed  Google Scholar  * Taylor, L. H., Welburn, S. C. & Woolhouse, M. E. Theileria annulata: virulence and transmission from

single and mixed clone infections in cattle. _Exp. Parasitol._ 100(3), 186–195 (2002). Article  PubMed  Google Scholar  * Al-Fahdi, A. _Molecular Identification and Phylogenetic Studies of

Theileria Parasite in Oman_. 2015, Sultan Qaboos university. * Tageldin, M. H., Fadiya, A.A.-K., Sabra, A.A.-Y. & Ismaily, S.I.A.-I. Theileriosis in sheep and goats in the Sultanate of

Oman. _Trop. Anim. Health Prod._ 37(6), 491–493 (2005). Article  CAS  PubMed  Google Scholar  * Menegon, M. _et al._ Frequency distribution of antimalarial drug resistance alleles among

plasmodium falciparum isolates from Gezira State, Central Sudan, and Gedarif State, Eastern Sudan. _Am. J. Trop. Med. Hygiene_ 83(2), 250–257 (2010). Article  Google Scholar  * Bashey, F.

Within-host competitive interactions as a mechanism for the maintenance of parasite diversity. _Philos. Trans. R. Soc. B Biol. Sci._ 370(1675), 20140301 (2015). Article  Google Scholar  *

Leemans, I., Hooshmand-Rad, P. & Uggla, A. The indirect fluorescent antibody test based on schizont antigen for study of the sheep parasite _Theileria lestoquardi_. _Vet. Parasitol._

69(1–2), 9–18 (1997). Article  CAS  PubMed  Google Scholar  * Goh, S. _et al._ Identification of Theileria lestoquardi antigens recognized by CD8+ T cells. _PLoS ONE_ 11(9), e0162571 (2016).

Article  PubMed  PubMed Central  Google Scholar  * Morrison, W. I. Progress towards understanding the immunobiology of Theileria parasites. _Parasitology_ 136(12), 1415–1426 (2009). Article

  Google Scholar  * Leemans, I., Brown, D., Hooshmand-Rad, P., Kirvar, E. & Uggla, A. Infectivity and cross-immunity studies of _Theileria lestoquardi_ and _Theileria annulata_ in sheep

and cattle: I. In vivo responses. _Veterinary Parasitol._ 82(3), 179–192 (1999). Article  CAS  Google Scholar  * Pacheco, M. A. _et al._ Evidence for negative selection on the gene encoding

rhoptry-associated protein 1 (RAP-1) in Plasmodium spp. _Infect. Genet. Evol._ 10(5), 655–661 (2010). Article  CAS  PubMed  Google Scholar  * Mideo, N. Parasite adaptations to within-host

competition. _Trends Parasitol._ 25(6), 261–268 (2009). Article  PubMed  Google Scholar  * Bruce, M. C. _et al._ Cross-species interactions between malaria parasites in humans. _Science_

287(5454), 845–848 (2000). Article  ADS  CAS  PubMed  Google Scholar  * Morrison, W. & McKeever, D. Current status of vaccine development against Theileria parasites. _Parasitology_

133(S2), S169–S187 (2006). Article  PubMed  Google Scholar  * Awad, H. _et al._ _Theileria lestoquardi_ in Sudan is highly diverse and genetically distinct from that in Oman. _Infect. Genet.

Evol._ 62, 46–52 (2018). Article  PubMed  Google Scholar  * Smith, T., Felger, I., Kitua, A., Tanner, M. & Beck, H.-P. Dynamics of multiple Plasmodium falciparum infections in infants

in a highly endemic area of Tanzania. _Trans. R. Soc. Trop. Med. Hygiene_ 93(1), 35–39 (1999). Article  Google Scholar  * Staroscik, A., dsDNA copy number calculator_. URI Genomics &

Sequencing Center_, (2004). * Burnham, K. P. & Anderson, D. R. _Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach_ (Springer Science & Business

Media, Berlin, 2003). MATH  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to the farmers and the staff of the Ministry of Agriculture and Fisheries, Oman, for their

support with field surveys. We appreciate the support of the technical staff of the Biochemistry Department, Sultan Qaboos University, Oman. This research is supported by Gulf States

Cooperation Council grant (GCC)CL/SQU-GCC/17/01. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Biochemistry, College of Medicine and Health Sciences, Sultan Qaboos University,

AlKhoud 123, Muscat, Oman Hoyam Awad, Milagros Postigo & Hamza A. Babiker * Division of Population Medicine, School of Medicine, College of Biomedical Sciences, Cardiff University,

Cardiff, UK Amal A. H. Gadalla * Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman Salama Al-Hamidhi * College of Agriculture and Marine Sciences, Sultan

Qaboos University, Muscat, Oman Mohammed H. Tageldin & Eugene H. Johnson * Department of Microbiology and Immunology, Weill Cornell Medicine - Qatar, Cornell University, Qatar

Foundation, Doha, Qatar Sini Skariah & Ali A. Sultan * Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of

Glasgow, Glasgow, UK Brian Shiels * Biological and Environmental Science and Engineering Diversion, King Abdullah, University for Science and Technology, Thuwal, Saudi Arabia Arnab Pain *

GI-CoRE, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan Arnab Pain * Institute of Immunology and Infection Research, School of Biological Sciences, University of

Edinburgh, Edinburgh, UK Joanne Thompson Authors * Hoyam Awad View author publications You can also search for this author inPubMed Google Scholar * Amal A. H. Gadalla View author

publications You can also search for this author inPubMed Google Scholar * Milagros Postigo View author publications You can also search for this author inPubMed Google Scholar * Salama

Al-Hamidhi View author publications You can also search for this author inPubMed Google Scholar * Mohammed H. Tageldin View author publications You can also search for this author inPubMed 

Google Scholar * Sini Skariah View author publications You can also search for this author inPubMed Google Scholar * Ali A. Sultan View author publications You can also search for this

author inPubMed Google Scholar * Eugene H. Johnson View author publications You can also search for this author inPubMed Google Scholar * Brian Shiels View author publications You can also

search for this author inPubMed Google Scholar * Arnab Pain View author publications You can also search for this author inPubMed Google Scholar * Joanne Thompson View author publications

You can also search for this author inPubMed Google Scholar * Hamza A. Babiker View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.A., S.H.,

M.P., M.H.T., A.H.G.: Experimental laboratory and data analysis. A.P.: Provided materials. H.A.B., J.T., E.J., B.S., A.A.S., S.S., A.P.: Conceived and developed idea. H.B., J.T., B.S.:

Manuscript preparation. CORRESPONDING AUTHOR Correspondence to Hamza A. Babiker. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

PUBLISHER'S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1.

SUPPLEMENTARY LEGEND. RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation,

distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and

indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit

line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE Awad, H., Gadalla, A.A.H., Postigo, M. _et al._ Dynamics and within-host interaction of _Theileria lestoquardi_ and _T. ovis_ among naive sheep in Oman. _Sci Rep_

10, 19802 (2020). https://doi.org/10.1038/s41598-020-76844-2 Download citation * Received: 24 December 2019 * Accepted: 22 October 2020 * Published: 13 November 2020 * DOI:

https://doi.org/10.1038/s41598-020-76844-2 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative