The emergence of coinfection with Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia, and Trypanosoma evansi is an important problem that endangers human health, animal quality and public sanitary security. These pathogenic protozoans play important roles in establishment of similar clinical signs of diseases in humans, pigs, sheep, and rabbits, including fever, diarrhea, hepatitis, encephalitis, and reproductive disorders. Therefore, a rapid and specific diagnostic method to simultaneously detect these five pathogens is urgently required. Here, we developed a TaqMan-probe-based quantitative real-time polymerase chain reaction (qPCR) for the simultaneous detection of these five pathogens for the first time. Specific primers and probes were designed targeting the G3PDH gene of Toxoplasma gondii, the NC5 gene of Neospora caninum, the ADF gene of Eimeria stiedai, the GDH gene of Giardia lamblia, the COX1 gene of Trypanosoma evansi, and a TaqMan-probe-based pentaplex qPCR assay capable of simultaneously detecting these five pathogens was developed. The assay showed strong specificity, with no cross-reactivity detected against nucleic acids from other control pathogens. The assay demonstrated high sensitivity, with the lower limit of quantification (LLOQ) of 10 copies per reaction for the recombinant plasmid standards pTgG3PDH, pNcNC5, pEsADF, pGlGDH, pTeCOX1, and the limit of detection (LOD) as low as 1.1 copies. The standard curves exhibited excellent linearity (correlation coefficient values of 0.996, 0.996, 0.996, 0.992, and 0.996, respectively) and high amplification efficiencies (95.534%, 96.203%, 107.818%, 100.851%, and 104.487%, respectively). The assay also exhibited excellent repeatability and reproducibility, with inter- and intra-assay coefficient of variation (CV) ranging from 0.07% to 2.13%. The anti-interference test showed that high-concentration nucleic acids did not interfere with low-concentration nucleic acids, thus ensuring excellent detection results even in complex samples. Among 210 clinical samples, the assay detected Toxoplasma gondii in 2.38%, Neospora caninum in 2.38%, Eimeria stiedai in 10%, and Giardia lamblia in 36.19%. The coinfection rates were 8.1% for Eimeria stiedai & Giardia lamblia. This TaqMan-probe-based pentaplex qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia, and Trypanosoma. It is of great significance to safeguard human health.

Pre-exposure prophylaxis of HIV infection with a microbicide is one of the most effective ways to block HIV/AIDS transmission. In this study, a potent and long-acting HIV entry inhibitor peptide 2P23 was developed as a novel HIV microbicide, and its toxicity and safety were evaluated through animal model in vivo for the first time, which provided a new effective means for the prevention of HIV/AIDS. In order to evaluate the potential effect to cause human toxicity and possible health hazards likely to arise from repeated rectovaginal application of 2P23, we conducted an acute toxicity in which a dose of 300 mg/kg of 2P23 was administrated by rectovaginal three times within 24-hour in male and female Sprague Dawley rats. Furthermore, a 90-day repeated rectovaginal treatment tests in rats were used to evaluate the chronic and reproductive toxicity of 50, 100, 200 mg/kg of 2P23. The effects of 2P23 administration on morbidity, mortality, clinical signs, changes in body weight, food consumption, clinical pathology, organ weight, hematological and physiological biochemical parameter indicators, histopathology, changes in rectovaginal microbiota, fertility, and early embryonic development were assessed. There were no mortality or abnormal clinical signs, no significant changes in body weight gain or hematological and physiological biochemical parameter indicators, no changes in necropsy and histopathology findings of vital organs, and no significant changes in rectum and vagina microbiota associated with the administration of 2P23. There were no effects on fertility and early embryonic development in male and female rats. The results indicate that 2P23 does not cause significant adverse effects in the 90-day and suggest that the no observed adverse effect level is 300 mg. This study has important application value and good innovation for the development of anti-HIV agents for rectovaginal administration. The novel HIV microbicide is safe and effective for the prevention and control of HIV/AIDS sexually transmitted infections, which enriches people’s understanding of the novel HIV microbicide gel. The study has important application value and good innovation for the development of anti-HIV agents for rectovaginal administration.

The emergence of coinfection with Toxoplasma gondii (Tg), Neospora caninum (Nc), Eimeria stiedai (Es), Giardia lamblia (Gl), and Trypanosoma evansi (Te) is an important problem that endangers human health, animal quality and public sanitary security. These pathogenic protozoans play important roles in the establishment of similar clinical signs of diseases in human/sheep/pig/rabbits, including diarrhea, hepatitis, encephalitis, and reproductive disorders. Therefore, a rapid and specific diagnostic method to simultaneously detect Tg, Nc, Es, Gl, and Te is urgently required. Here, we developed a TaqMan-probe-based quantitative real-time polymerase chain reaction (qPCR) for rapid detection of Tg, Nc, Es, Gl, and Te for the first time. Specific primers and TaqMan-probes were designed for the conserved regions in the G3PDH region of Tg, NC5 region of NC, ADF region of Es, GDH region of Gl, and COX1 region of Te, and pentaplex qPCR for simultaneous detection of Tg, Nc, Es, Gl and Te was developed. The assay showed a strong specificity for detecting Tg, Nc, Es, Gl and Te, and had no cross-reactivity with other control pathogenic parasites. The assay had high sensitivity with the lower limit of quantification (LLOQ) and the limit of detection (LOD) are 10 and 3 copies per reaction. The assay exhibited excellent repeatability and reproducibility of the intra-assay coefficient of variation (CV) of 0.07–2.10% and the inter-assay CV of 0.22–2.13%. Finally, the developed TaqMan-probe-based pentaplex qPCR was used to detect 200 clinical samples. The results indicated that the positive detection rate of Tg, Nc, Es, Gl were 2.5% (5/200), 2.5% (5/200), 5.5% (11/200), 33% (66/200), respectively, and the result showed good consistency with conventional PCR. The developed TaqMan-probe-based pentaplex qPCR assay has the advantages of high sensitivity and strong specificity, high throughput, convenient and fast, and cost-effectiveness. It is of great significance to safeguard animal quality and human health.

Cryptosporidium parvum (Cp), Strongyloides stercoralis (Ss), Trypanosoma cruzi (Tc), Theileria ovis (To) and Echinococcus granulosus (Eg) are significant in causing similar clinical symptoms in both humans and animals. These symptoms include diarrhea, fever, pneumonia, hepatitis, encephalitis, cardiovascular, and gastrointestinal diseases, as well as reproductive system diseases. Therefore, it is urgent to establish a rapid, specific, and sensitive method to simultaneously detect these five pathogens. Here, we develop a TaqMan probe-based pentaplex real-time fluorescence quantitative PCR (qPCR) for simultaneous detection of Cp, Ss, Tc, To, and Eg for the first time. Five sets of specific primers and probes were designed. The 5′ end of five probes were labeled with five different fluorescent groups (FAM, VIC, ABY, JUN, CY5) that do not interfere with each other, while the 3′ end were labeled with quenching groups (MGB-NFQ, QSY). The instrument collects fluorescence signals at different wavelengths in five independent optical channels in qPCR to achieve simultaneous specific amplification and quantitative detection of five targets within one tube. A five-color qPCR was developed, which can rapidly and quantitatively distinguish and identify Cp, Ss, Tc, To, and Eg in a single tube. This method shows strong specificity and no cross-reaction with other control pathogens. This method has high sensitivity, with the LLOQ of 10 copies and the LOD as low as 1 copy. The standard curve shows a good linear relationship. The amplification efficiency is high. The repeatability and reproducibility are excellent. The interference test shows that high concentrations of nucleic acids do not interfere with low concentrations of nucleic acids, and can ensure excellent detection results even in complex samples for 1.5 hours. A total of 246 clinical samples were detected: 4.88% were positive for Cp, 2.44% for Ss, and 15.85% for To, while 0.81% for co-infections of Cp/To. This TaqMan probe-based pentaplex qPCR enables rapid detection and differential identification of five pathogens from clinical samples, eliminating the need for multiple single-plex qPCR. It can potentially assist clinicians in diagnosis and treatment planning by rapidly detecting pathogens in clinical samples and further quantitative analysis. It is of extremely significant importance for protecting human health.