Theileria orientalis Ikeda in Cattle, Alabama, USA

Simple Summary Theileria orientalis Ikeda poses significant challenges to the cattle industry, encompassing economic repercussions, trade-related issues, and constraints regarding control and treatment measures. This parasite can precipitate a range of adverse effects in cattle, including diminished milk production, weight loss, anemia, and, in severe instances, cattle fatalities. These adverse impacts directly translate into economic burdens for farmers and the broader cattle industry. The financial costs associated with treating afflicted animals and implementing control strategies can be substantial. Additionally, occurrences of Theileria orientalis Ikeda in regions where it is not endemic can lead to trade restrictions and impediments affecting both cattle and cattle products. This can curtail the international movement of cattle and influence global trade in livestock. Furthermore, there is a dearth of effective treatment options for infected cattle, and severe cases frequently culminate in fatalities. This lack of viable treatment exacerbates the difficulties in disease management. Addressing the presence of Theileria orientalis Ikeda in the USA necessitates comprehensive efforts to understand the prevalence and distribution of the pathogen. This study utilizing PCR followed by DNA sequencing identified two cases of Theileria orientalis Ikeda-positive cattle, marking the first report of this virulent genotype in Alabama, USA. Abstract Theileria orientalis Ikeda genotype, a parasite causing a disease in cattle that leads to significant economic challenges in Asia, New Zealand, and Australia, has been identified in seven U.S. States since 2017. Two previously validated PCR tests for Theileria followed by DNA sequencing were performed to test blood samples collected from 219 cattle in Alabama, USA, during the period of 2022–2023. Bidirectional Sanger sequencing revealed that the MPSP gene sequences (639–660 bp) from two cattle in Lee and Mobile Counties of Alabama exhibited a 100% match with those of recognized T. orientalis Ikeda strains, and showed similarities ranging from 76% to 88% with ten other T. orientalis genotypes. A high copy number of T. orientalis Ikeda was detected in the blood of infected cattle (ALP-1: 1.7 × 105 and 1.3 × 106/mL whole blood, six months apart; ALP-2: 7.1 × 106/mL whole blood). Although the confirmed competent vector for T. orientalis Ikeda, Haemaphysalis longicornis tick, has not yet been identified in Alabama, the persistent nature of T. orientalis Ikeda infection and the detection of a high pathogen burden in seemingly healthy cattle in this study suggest that other tick species, as well as shared needles and dehorning procedures, could facilitate pathogen transmission within the herd. Continued investigations are necessary for the surveillance of T. orientalis Ikeda and Haemaphysalis longicornis ticks in Alabama and other U.S. states, along with assessing the pathogenicity of T. orientalis Ikeda infections in cattle.

To assess the T. orientalis status in cattle, sheep, and goats in Alabama, USA, we conducted PCR assays followed by DNA sequencing analysis on existing convenience samples of whole blood and blood samples obtained from farms in Alabama.

Whole Blood Samples
Samples for analysis came from two sources.Whole-blood samples in EDTA from cattle (n = 72) presenting to the Auburn University Large Animal Teaching hospital between September 2022 and August 2023 for regular physical examination and the diagnosis and treatment of diseases where whole blood was submitted to the Clinical Pathology Laboratory were used in the study.Aliquots of the whole-blood samples were used for routine complete blood counts and biochemical profiles.Additionally, 147 bovine blood samples from three cattle farms associated with Auburn University College of Veterinary Medicine located in Marion County (n = 50), Lee County (n = 33), and Tallapoosa County (n = 64) of Alabama were also used to screen Theileria in this study.The protocol for the collection of bovine samples was reviewed and approved by the Institutional Animal Care and Use Committee of Auburn University (IACUC #2022-5112).
Blood samples were sent to Auburn at ambient temperature.The High-Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) was used to extract DNA from 400 µL aliquots as published [13,14].The extracted DNA was eluted in 200 µL elution buffer, and was preserved at −80 • C until PCR was performed in this study.

Detection of Theileria and Anaplasma DNA by PCRs
Two previously validated quantitative PCRs, an MPSP gene-based Theileria TaqMan PCR [15] and a FRET-PCR targeting the rRNA of Theileria [14], were used to detect Theileria DNA in this study.The primers and probes as well as the primers to sequence the MPSP gene are shown in Table 1.
The products of Theileria-positive PCRs were sent to ELIM Biopharmaceuticals (Hayward, CA, USA) for Bidirectional Sanger sequencing.The nucleotide sequences were submitted to NCBI to obtain GenBank Accession numbers (OR570618, OR570619), and a phylogenetic tree was generated to compare the nucleotide sequences of Theileria identified in this study with those of all recognized 11 T. orientalis genotypes (Figure 1).  of T. orientalis, were concatenated and aligned using CLUSTALW.A maximum likelihood phylogenetic tree was generated employing a p-distance model.Bootstrap values are expressed as percentages based on 1000 replications, and the bar represents evolutionary distances as 0.05 changes per nucleotide position.The genotypes of T. orientalis, along with their GenBank accession numbers, are provided.Notably, the sequences of T. orientalis Ikeda identified in this study (ALP-1: OR570618; ALP-2: OR570619) exhibited 100% identity with those of recognized T. orientalis Ikeda strains, while demonstrating 76-88% similarity with other T. orientalis genotypes.
Table 1.Primers and probes used in this study.

PCR and the Target Gene
Oligonucleotides References The Theileria-positive samples identified in this study were submitted to the Molecular diagnostic laboratory at Auburn University College of Veterinary Medicine for the detection of Anaplasma DNA following the published protocol [16].

Results
Both the MPSP-based TaqMan PCR and rRNA-targeting FRET-PCR, followed by DNA sequencing, successfully identified T. orientalis DNA in 2 out of 89 convenience blood samples.However, none of the 147 bovine blood samples from three different farms tested positive for T. orientalis.Notably, the two T. orientalis-positive samples were found to be free of Anaplasma spp.
In Lee County, Alabama, a one-year-old female heifer (ALP-1 with an accession number OR570618) was found to carry T. orientalis Ikeda DNA.This heifer exhibited normal red blood cell (RBC) and hemoglobin (HGB) counts but displayed elevated monocyte counts (2.3/µL) and white blood cell counts (2.1 × 10 3 /µL).Additionally, increased levels of glucose (313 mg/dL) were observed, alongside low iron levels (25 µg/dL).This heifer was being treated for an acute free gas bloat resulting from grain overload.She recovered from this episode of ruminal acidosis following treatment and has been a healthy member of the resident herd since discharge.The initial test revealed the presence of 1.7 × 10 5 T. orientalis Ikeda/mL in whole blood, and a follow-up test six months later showed the increased count of 1.3 × 10 6 /mL in whole blood.
A four-month-old male calf from Mobile County, Alabama, presented with fever, swollen lymph nodes, anorexia, pyrexia, cough, dyspnea, lethargy, tachypnea, polyuria, and diarrhea.Bloodwork, including chemistry and a complete blood count, revealed no significant abnormalities except for decreased fibrinogen levels, indicating recovery from a previous inflammatory event.A urinalysis was also performed, revealing dilute urine but no signs of infection or inflammation of the bladder.This calf tested positive for T. orientalis Ikeda (ALP-2 with an accession number OR570619), with a copy number of 7.1 × 10 6 /mL in whole blood.

Discussion
T. orientalis Ikeda is a protozoan parasite of cattle that has significant implications for the livestock industry.In this study, we reported for the first time the identification of this virulent genotype Ikeda in the cattle from Alabama, USA.Persistent infection with different variants of T. orientalis is a common occurrence [7,18], and the immune mechanisms responsible for disease resistance are not yet fully understood.In this study, a follow-up test conducted six months later revealed an increased count of 1.3 × 10 6 /mL of T. orientalis Ikeda in the whole blood of one animal, compared to the initial count of 1.7 × 10 5 T. orientalis Ikeda/mL whole blood, with both cattle appearing to be in relatively good health, and no evidence of anemia or fever being observed.This supports the notion that T. orientalis can lead to chronic and persistent infections, emphasizing the need for further research into the pathogenicity of T. orientalis Ikeda infections in ruminants.
An experimental transmission trial conducted by the USDA's Agricultural Research Service (ARS) in collaboration with the Virginia Tech Animal Laboratory Services (ViTALS) laboratory has confirmed the vector competence of the Haemaphysalis longicornis tick (ALHT: Asian long-horn tick) for T. orientalis Ikeda [17].ALHT can feed on a wide range of wildlife and domestic species, including birds, white-tailed deer, companion animals, livestock, equines, and humans.As of September 2023, ALHT has been identified in 18 states [19].Once introduced into suitable habitats, ALHT populations can proliferate rapidly, partly due to the exhibition of multiple genetic types, including both parthenogenetic and bisexual reproduction [20,21].
Although ALHT has not yet been identified in Alabama, given the persistent nature of T. orientalis Ikeda infection and the detection of a high burden of T. orientalis Ikeda in apparently healthy cattle in this study, it is plausible that other tick species, as well as shared needles and dehorning procedures, could facilitate the spread of the pathogen within the herd.Telionis et al. reported an 8.7% (172/1980) prevalence of genotype Ikeda in Virginia Market Cattle, 2018-2020 [6].It is likely that T. orientalis Ikeda will be identified in many other states and become established throughout the USA.

Conclusions
In conclusion, PCR tests followed by DNA sequencing confirmed the presence of T. orientalis Ikeda DNA in the blood of two cattle in Alabama, USA.Continued research in epidemiology, vector ecology and vaccine development will contribute to a better understanding of Theileria orientalis Ikeda and help develop more effective strategies for its control and prevention, benefiting both the cattle industry and animal health.Informed Consent Statement: Informed consent was waived as this study utilized convenient and anonymous diagnostic samples.

Figure 1 .
Figure 1.Phylogenetic tree displaying major piroplasm surface protein (MPSP) gene sequences for Theileria orientalis.The 739-760-bp nucleotide sequences of the MPSP gene, representing 11 genotypes of T. orientalis, were concatenated and aligned using CLUSTALW.A maximum likelihood

Funding:
This research was supported, in part, by the Molecular Diagnostic Laboratory at Department of Pathobiology Auburn University.Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Care and Use Committee of Auburn University (IACUC #2022-5112 approved on 25 October 2022).