Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R² = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

Background: Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. Results: A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. Conclusions: Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

To validate the prevalence and biodiversity of ticks and tick-borne pathogens in Chongqing, a total of 601 ticks were collected from dogs, cattle, and goats within the Ta-pa Mountain range in Chongqing, China. Five distinct tick species were identified, including Ixodes ovatus (1.66%, 10/601), I. acutitarsus (0.50%, 3/601), Haemaphysalis flava (10.32%, 62/601), Ha. hystricis (9.82%, 59/601), and Ha. longicornis (77.70%, 467/601). A suit of semi-nest PCR and nest PCR primers were custom-synthesized for the detection of tick-borne pathogens. The analysis yielded positive results for 7.15% Rickettsia (Candidatus R. principis, R. japonica, and R. raoultii), 3.49% Anaplasma (A. bovis and A. capra), 1.16% Ehrlichia, 1.83% Coxiella burnetii, and 3.49% protozoa (Theileria. capreoli, T. orientalis, T. luwenshuni, and Babesia sp.) in ticks. Notably, Ca. R. principis was identified for the first time in I. ovatus and Ha. longicornis. These findings underscore the significant prevalence and diversity of ticks and their associated pathogens within the Chongqing Ta-pa Mountain region. This study accordingly provides an extensive dataset that contributes to the epidemiological understanding and disease prevention strategies for tick-borne illnesses in the local area.