ObjectiveTo explore the application effects of environmentally friendly reagents versus traditional reagents in tissue processing， paraffin sectioning， hematoxylin-eosin （HE） staining， special staining， and immunohistochemistry. MethodsPancreatic， renal， splenic， hepatic， pulmonary， skeletal muscle， and cardiac tissues were collected from mice and New Zealand rabbits， and randomly divided into a traditional group （Group A）， an environmentally friendly reagent I group （Group B）， and an environmentally friendly reagent Ⅱ group （Group C）. The processed tissue sections from all three groups were subjected to paraffin sections， HE staining， phosphotungstic acid-hematoxylin staining， and Mallory trichrome staining. The pancreatic tissue sections were subjected to immunohistochemical detection of insulin protein. Evaluation criteria included tissue morphological integrity， embedding operability， section quality， and staining effects. ResultsNo significant differences were observed among the three groups of samples in terms of fixation efficacy， embedding operations， and section quality. After HE staining， all groups exhibited bright nuclear and cytoplasmic staining with clear contrast， with excellent-and-good rates of 98%， 99%， and 97% for Groups A， B， and C， respectively， showing no statistically significant differences （A vs. B， P=0.561； A vs. C， P=0.651）. In phosphotungstic acid-hematoxylin staining， Groups A and C showed clear structural contrast and bright staining for striated muscle and collagen fibers， whereas Group B exhibited uneven staining. The excellent-and-good rates were 100%， 10%， and 95% for Groups A， B， and C， respectively， with significant differences between Group A and Group B （P＜0.01） but no statistical difference between Group A and Group C （P=0.311）. In Mallory trichrome staining， Groups A and B outperformed Group C. The excellent-and-good rates were 95%， 90%， and 15% for Groups A， B， and C， respectively， with significant differences between Group A and Group C （P＜0.01）， but no statistical difference between Group A and Group B （P=0.548）. Immunohistochemical results showed that Group A had a clean background with no non-specific staining， while Groups B and C exhibited significant non-specific staining， with statistically significant differences in non-specific staining rates between the groups （A vs. B， 5% vs. 100%， P＜0.01； A vs. C， 5% vs.100%， P＜0.01）. ConclusionIn HE staining， environmentally friendly reagents demonstrated effective tissue fixation and staining results comparable to traditional reagents， meeting the teaching requirements. For special staining， Group B and Group C performed better in Mallory trichrome staining and phosphotungstic acid-hematoxylin staining， respectively. However， environmentally friendly reagents exhibited non-specific staining issues in immunohistochemistry， where further optimization is required.

Strangles,caused by Streptococcus equi subsp.equi,remains one of the most prevalent and high-incidence infectious diseases in intensive donkey farms,posing a significant threat to the healthy development of the donkey industry.Vaccination serves as an effective measure for the pre-vention and control of the disease,however,there is currently no attenuated vaccine against this disease in China.To provide a candidate strain for the development of a live attenuated strangles vaccine,this study focused on a wild-type S.equi subsp.equi strain isolated from donkeys.Using homologous recombination gene knockout technology,the aroA gene(encoding 5-enolpyru-vylshikimate-3-phosphate synthase)and the sag A gene(encoding the precursor of streptolysin S toxin)were sequentially deleted to construct a double-gene-deletion strain(ΔsagA/aroA).The virulence and key biological characteristics of the mutant strain were systematically evaluated.TheΔsagA/aroA strain was successfully generated,exhibiting complete loss of hemolytic activity and maintaining stable genetic inheritance over 60 consecutive passages.Electron microscopy revealed that the mutant retained morphological characteristics compared to the wild-type strain,and its growth rate was significantly slower(P＜0.000 1).Virulence assessment using a challenge dose of 1× 105 CFU/0.2 mL(the minimum fully lethal dose of the wild-type strain)demonstrated markedly attenuated virulence in the mutant.Immunization trials with 1 ×104 CFU/0.2 mL of theΔsagA/aroA strain revealed a increase in ELISA antibody titers by day 7 post-vaccination,and higher levels at days 14 and 21.Notably,antibody levels in the experimental group were significant-ly higher than those in the control group(P＜0.000 1).These findings confirm that the double-gene-deletion strain S.equi subsp.equi ΔsagA/aroA exhibits reduced virulence while retaining im-munogenicity,which suggested it can be used as a promising candidate strain for further develop-ment of a live attenuated strangles vaccine.

Strangles,caused by Streptococcus equi subsp.equi,remains one of the most prevalent and high-incidence infectious diseases in intensive donkey farms,posing a significant threat to the healthy development of the donkey industry.Vaccination serves as an effective measure for the pre-vention and control of the disease,however,there is currently no attenuated vaccine against this disease in China.To provide a candidate strain for the development of a live attenuated strangles vaccine,this study focused on a wild-type S.equi subsp.equi strain isolated from donkeys.Using homologous recombination gene knockout technology,the aroA gene(encoding 5-enolpyru-vylshikimate-3-phosphate synthase)and the sag A gene(encoding the precursor of streptolysin S toxin)were sequentially deleted to construct a double-gene-deletion strain(ΔsagA/aroA).The virulence and key biological characteristics of the mutant strain were systematically evaluated.TheΔsagA/aroA strain was successfully generated,exhibiting complete loss of hemolytic activity and maintaining stable genetic inheritance over 60 consecutive passages.Electron microscopy revealed that the mutant retained morphological characteristics compared to the wild-type strain,and its growth rate was significantly slower(P＜0.000 1).Virulence assessment using a challenge dose of 1× 105 CFU/0.2 mL(the minimum fully lethal dose of the wild-type strain)demonstrated markedly attenuated virulence in the mutant.Immunization trials with 1 ×104 CFU/0.2 mL of theΔsagA/aroA strain revealed a increase in ELISA antibody titers by day 7 post-vaccination,and higher levels at days 14 and 21.Notably,antibody levels in the experimental group were significant-ly higher than those in the control group(P＜0.000 1).These findings confirm that the double-gene-deletion strain S.equi subsp.equi ΔsagA/aroA exhibits reduced virulence while retaining im-munogenicity,which suggested it can be used as a promising candidate strain for further develop-ment of a live attenuated strangles vaccine.

Objective:To investigate the clinical characteristics and pregnancy outcomes in pregnant women with left ventricular non-compaction (LVNC).Methods:The clinical data of seven pregnant women with LVNC from January 2011 to December 2021 in Beijing Anzhen Hospital,Capital Medical University were retrospectively analyzed, including age, gestational age of symptom first occured, LVNC history, clinical symptoms, New York Heart Association (NYHA) cardiac function class, echocardiography, blood brain natriuretic peptide (BNP), treatment and the maternal and fetal outcomes.Results:Five cases were diagnosed before pregnancy, of which there were three women with medication; one case diagnosed in the month of pregnancy; one case diagnosed at 36 weeks of gestation. NYHA cardiac function was grade Ⅰ in four cases and grade Ⅱ in three cases before or during the first trimester of pregnancy. Of the five pregnant women who underwent echocardiography, there were one case of left ventricular insufficiency, three cases of mild left ventricular dysfunction and one case of normal left ventricular function before or during the first trimester of pregnancy. Of the five pregnant women to the second and third trimester of pregnancy, there were one case of grade Ⅳ, one case of grade Ⅲ, two cases of grade Ⅱ-Ⅲ and one case of grade Ⅱ in NYHA class ; three cases of left ventricular insufficiency, two cases of normal left ventricular function by echocardiography four cases had cardiac symptoms at 15-24 weeks of gestation and were treated with medication. In four cases, blood BNP increased to 214-1 197 ng/L during pregnancy, and were 89-106 ng/L after termination of pregnancy. There were 4 cases with arrhythmia. Indications for termination of pregnancy: LVNC complicated with heart failure in two cases, LVNC complicated with decreased cardiac function and threatened preterm birth in one case, complicated with pregnancy at full term in two cases, LVNC complicated with severe pulmonary hypertension in one case, and left ventricular dysfunction in one case. Cesarean section in four cases in the third-trimester, in one case in the second-trimester, and forceps curettage in two cases were taken. Two full-term infants,two preterm infants were born without LVNC.Conclusions:Women diagnosed with LVNC and low left ventricular ejection fraction before pregnancy are more prone to decreased cardiac function during pregnancy. Carrying out pregnancy risk assessment and strengthening the multi-disciplinary team management of high risk factors in pregnancy are conducive to achieve good pregnancy outcomes.