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Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines., (© 2024. The Author(s).)

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 10 4 copies/µl = A. marginale, 5.04 × 10 6 copies/µl = A. ovis, and 4.58 × 10 3 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 10 3 copies/µl of A. marginale, 5.04 × 10 3 copies/µl of A. ovis, 4.58 × 10 3 copies/µl of A. phagocytophilum, and 5.51 × 10 3 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited., (© 2021. The Author(s).)

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis (HGA) is an obligate intracellular Gram-negative bacterium with the genome size of 1.47 megabases. The intracellular life style and small size of genome suggest that A. phagocytophilum has to modulate a multitude of host cell physiological processes to facilitate its replication. One strategy employed by A. phagocytophilum is through its type IV secretion system (T4SS), which translocates bacterial effectors into target cells to disrupt normal cellular activities. In this study we developed a TEM-1 β-lactamase based protein translocation assay and applied this assay for identification of A. phagocytophilum T4SS effectors. An A. phagocytophilum hypothetical protein, APH0215 is identified as a T4SS effector protein and found interacting with trans-Golgi network in transfected cells. Hereby, this protein translocation assay developed in this study will facilitate the identification of A. phagocytophilum T4SS effectors and elucidation of HGA pathogenesis.