The gene for the 32 kDa surface protein (p32) of Theileria sergenti was cloned into lambda gt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected the Pst I-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and the transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

Numbers of patients with coronavirus disease 2019 (COVID-19) have increased rapidly worldwide. Plasma levels of full-length galectin-9 (FL-Gal9) and osteopontin (FL-OPN) as well as their truncated forms (Tr-Gal9, Ud-OPN, respectively), are representative inflammatory biomarkers. Here, we measured FL-Gal9, FL-OPN, Tr-Gal9, and Ud-OPN in 94 plasma samples obtained from 23 COVID-19-infected patients with mild clinical symptoms (CV), 25 COVID-19 patients associated with pneumonia (CP), and 14 patients with bacterial infection (ID). The four proteins were significantly elevated in the CP group when compared with healthy individuals. ROC analysis between the CV and CP groups showed that C-reactive protein had the highest ability to differentiate, followed by Tr-Gal9 and ferritin. Spearman’s correlation analysis showed that Tr-Gal9 and Ud-OPN but not FL-Gal9 and FL-OPN, had a significant association with laboratory markers for lung function, inflammation, coagulopathy, and kidney function in CP patients. CP patients treated with tocilizumab had reduced levels of FL-Gal9, Tr-Gal9, and Ud-OPN. It was suggested that OPN is cleaved by interleukin-6-dependent proteases. These findings suggest that the cleaved forms of OPN and galectin-9 can be used to monitor the severity of pathological inflammation and the therapeutic effects of tocilizumab in CP patients.

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.

A pair of synthetic oligonucleotide primers, designed from the gene encoding a 32-kDa intraerythrocytic piroplasm surface protein of Theileria sergenti, were used to amplify parasite DNA from the blood of T. sergenti-infected cattle by means of the polymerase chain reaction (PCR). PCR-amplified DNA was examined by electrophoresis and by dot blot or microplate hybridization using a parasite-specific cDNA probe. PCR was specific for T. sergenti, since no amplification was detected with DNA from Anaplasma centrale, Babesia ovata, uninfected erythrocytes, and leukocytes. This method was sensitive enough to detect about 4.5 parasites per I.al of blood with a 10-p.l sample volume. Moreover, of 66 specimens from grazing cattle, 40 were microscopically positive, whereas PCR revealed that 54 samples were positive. Therefore, PCR provides a useful diagnostic tool for detecting T. sergenti-infected cattle, and it is significantly more sensitive than the current methods.