AbstractThis study aimed to assess the in vitro and in vivo disinfectant potential of peracetic acid (PAA) (1 mg/L) and hydrogen peroxide (H2O2) (20 mg/L) on the physicochemical and microbiological water quality parameters of fish aquaria, the microbial density of Nile tilapia muscular tissue, fish hepatic cortisol levels, and antioxidant biomarkers.

摘要本研究旨在评估过氧乙酸（PAA）（1 mg/L）和过氧化氢（H2O2）（20 mg/L）对鱼缸理化和微生物水质参数，尼罗罗非鱼肌肉组织的微生物密度，鱼肝皮质醇水平和抗氧化剂生物标志物的体外和体内消毒剂潜力。

In vitro, PAA and H2O2 reduced A. hydrophila colony viability by 5 log units after 30 and 5 min of contact time, respectively. PAA and H2O2 were added to aquaria water twice a week for the three-week experiment. Increased fish escape reflexes were observed only in the PAA group, which returned to normal within 10 min.

。在为期三周的实验中，每周两次将PAA和H2O2加入水族馆水中。仅在PAA组中观察到鱼类逃逸反射增加，该组在10分钟内恢复正常。

No mortalities were reported in either the PAA or H2O2 groups. An in vivo experimental challenge with a pathogenic strain of A. hydrophila revealed a 20% reduction in mortality in the PAA group, with no mortalities in the H2O2 group. Cortisol levels and antioxidant markers were measured to assess the impact of PAA and H2O2 on fish health.

PAA或H2O2组均未报告死亡。用嗜水气单胞菌致病菌株进行的体内实验挑战显示，PAA组死亡率降低了20%，H2O2组没有死亡率。测量皮质醇水平和抗氧化标记物以评估PAA和H2O2对鱼类健康的影响。

Cortisol levels in the PAA and H2O2 groups were significantly higher than in the control group after disinfectant exposure, but they progressively returned to normal. A significant reduction in superoxide dismutase (SOD) and catalase (CAT) activity, along with considerably higher glutathione peroxidase (GPx) and malondialdehyde (MDA) enzymatic activity, was observed in the PAA and H2O2 groups compared to the control group.

消毒剂暴露后，PAA和H2O2组的皮质醇水平显着高于对照组，但逐渐恢复正常。与对照组相比，在PAA和H2O2组中观察到超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性显着降低，以及谷胱甘肽过氧化物酶（GPx）和丙二醛（MDA）酶活性显着升高。

A substantial increase in total antioxidant capacity (TAC) was recorded in the PAA group. Physicochemical analyses revealed reduced pH and increased dissolved oxygen levels in the PAA and H2O2 groups. Microbiological analyses showed a significant reduction in bacterial density in water by 64% and 76% after 30 min of exposure to PAA and H2O2, respectively, w.

PAA组的总抗氧化能力（TAC）显着增加。物理化学分析显示PAA和H2O2组的pH值降低，溶解氧水平升高。微生物学分析显示，在暴露于PAA和H2O2 30分钟后，水中细菌密度分别显着降低64%和76%。

IntroductionBacterial disease outbreaks pose a significant challenge to freshwater and mariculture systems, causing up to a 50% loss in Oreochromis niloticus fish production in Egypt1,2,3. In recent years, Aeromonas species have been the primary cause of mass mortality outbreaks in aquaculture during the summer, leading to hemorrhagic septicemia4,5.

引言细菌性疾病的爆发对淡水和海水养殖系统构成了重大挑战，导致埃及尼罗罗非鱼产量损失高达50%1,2,3。近年来，气单胞菌是夏季水产养殖大规模死亡爆发的主要原因，导致出血性败血症4,5。

This opportunistic pathogen infects fish when a key factor disrupts the balance between pathogen, host, and environment. The heterogeneity of Aeromonas strains hinders vaccine development, making water disinfectants a viable alternative for preventing and controlling bacterial infections6.Maintaining preventive measures is crucial for managing fish diseases.

当关键因素破坏病原体，宿主和环境之间的平衡时，这种机会性病原体会感染鱼类。气单胞菌菌株的异质性阻碍了疫苗的开发，使水消毒剂成为预防和控制细菌感染的可行替代品6。维持预防措施对于管理鱼类疾病至关重要。

Several low-cost biosecurity approaches have proven effective and easy to implement in aquaculture, including systemic treatments with antibiotics, immunostimulants, feed additives, water disinfectants, or a combination of these measures7,8. Disinfectants and synthetic chemicals are widely used for disease prevention and control in fish and other aquatic species9.

几种低成本的生物安全方法已被证明在水产养殖中有效且易于实施，包括使用抗生素，免疫刺激剂，饲料添加剂，水消毒剂或这些措施的组合进行全身治疗7,8。消毒剂和合成化学品广泛用于鱼类和其他水生物种的疾病预防和控制9。

The disinfectant application frequency depends on the infection severity and water conditions10.Peracetic acid (PAA) and hydrogen peroxide (H2O2) are environmentally friendly disinfectants that decompose into harmless by-products, releasing no pollutants11. These agents are effective against various aquatic pathogens, with their microbicidal activity depending on concentration and ratio.

消毒剂的使用频率取决于感染的严重程度和水质状况10。过氧乙酸（PAA）和过氧化氢（H2O2）是环境友好的消毒剂，可分解成无害的副产物，不释放污染物11。这些药剂对各种水生病原体有效，其杀微生物活性取决于浓度和比例。

PAA is increasingly used in aquaculture as an antibacterial agent for therapeutic and disinfection purposes12,13. Unlike other disinfectants (formalin, chlorine, and ozone), PAA has a wide safety margin, lacks carcinogenic risks, produces negligible harmful by-products, and poses minimal health hazards14.

PAA越来越多地用于水产养殖，作为治疗和消毒目的的抗菌剂12,13。与其他消毒剂（福尔马林，氯和臭氧）不同，PAA具有广泛的安全边际，缺乏致癌风险，产生的有害副产物可忽略不计，对健康的危害最小14。

It rapidly dissociates.

它迅速分离。

In vitro bactericidal effect of PAA and H

PAA和H的体外杀菌作用

2

2

O

O

2

2

against

反对

A. hydrophila

A、 嗜水气单胞菌

Test conditionsThe bactericidal quantitative suspension test was adapted from BS EN 1276:2009. The test was conducted at room temperature (23–25 ºC) with 300 mg/L water hardness and a 5% organic load. Hard water was prepared by adding 972 mL of distilled water to 12 mL of Solution A (containing 19.84 g of anhydrous MgCl2/L and 46.24 g of anhydrous CaCl2/L) and 16 mL of Solution B (containing 35.02 g of NaHCO3/L).

测试条件杀菌定量悬浮液测试改编自BS EN 1276:2009。该测试在室温（23–25ºC）下进行，水硬度为300 mg/L，有机负荷为5%。通过向12 mL溶液A（含有19.84 g无水MgCl2/L和46.24 g无水CaCl2/L）和16 mL溶液B（含有35.02 g NaHCO3/L）中加入972 mL蒸馏水来制备硬水。

A 5% yeast extract solution was prepared by dissolving 50 g of yeast extract powder in 50 mL of distilled water. Then, 1 mL of this stock solution was added to the test suspension.Test suspension and test (N and N0)An A. hydrophila test (N) was conducted by adjusting the concentration of a subculture of the organism to 1.5–5 × 108 CFU/mL using the 0.5 McFarland standard and a solution of 0.1% tryptone and 0.85% NaCl in distilled water.

通过将50克酵母提取物粉末溶解在50毫升蒸馏水中来制备5%的酵母提取物溶液。然后，将1 mL该储备溶液加入到测试悬浮液中。测试悬浮液和测试（N和N0）嗜水气单胞菌测试（N）通过使用0.5 McFarland标准品和0.1%胰蛋白胨和0.85%NaCl的蒸馏水溶液将生物体传代培养物的浓度调节至1.5-5×108 CFU/mL来进行。

One milliliter of sterile 5% yeast extract was combined with 8 mL of fresh, diluted disinfectants and mixed thoroughly. The mixture was maintained at 20 °C ± 1 °C for 15 min in a water bath. Afterward, 1 mL of A. hydrophila suspension was added, mixed, and left for 5, 30, or 60 min of contact time. At the end of the contact time, 1 mL aliquots were taken and mixed with 8 mL of neutralization solution (containing 3.0 g/L lecithin, 30.0 g/L Tween 80, 30.0 g/L saponin, 5.0 g/L sodium thiosulfate, 1.0 g/L histidine, 1.0 g/L tryptone, and 8.5 g/L NaCl), followed by the addition of 1 mL of sterile distilled water.

将1毫升无菌5%酵母提取物与8毫升新鲜稀释的消毒剂混合，并充分混合。将混合物在水浴中保持在20℃±1℃15分钟。之后，加入1毫升嗜水气单胞菌悬浮液，混合，并放置5、30或60分钟的接触时间。在接触时间结束时，取1 mL等分试样并与8 mL中和溶液（含有3.0 g/L卵磷脂，30.0 g/L吐温80、30.0 g/L皂苷，5.0 g/L硫代硫酸钠，1.0 g/L组氨酸，1.0 g/L胰蛋白胨和8.5 g/L NaCl）混合，然后加入1 mL无菌蒸馏水。

The mixture was mixed well and left at 20 °C. After a 5-minute neutralization period, 1.0 mL of the neutralization solution was serially diluted to 108. Duplicate 1 mL aliquots of each dilution were plated on tryptic soy agar and incubated at 37 °C for 48 h. The microbial effect (ME) of the tested disinfectants was calculated.

将混合物充分混合并置于20℃。。将每种稀释液的重复1 mL等分试样铺在胰蛋白酶大豆琼脂上，并在37°C下孵育48小时。计算测试消毒剂的微生物效应（ME）。

A. hydrophila isolation and identificationPhenotypic characterizationColonies of A. hydrophila appeared as pinpoint yellow on Aeromonas agar base media and R-S media. Gram staining revealed gram-negative short motile bacilli. Biochemical reactions for oxidase and catalase tests were positive. Analytical profile indexing (API 20E) confirmed A.

A、 嗜水气单胞菌的分离和鉴定表型特征嗜水气单胞菌的菌落在气单胞菌琼脂基培养基和R-S培养基上显示为精确的黄色。革兰氏染色显示革兰氏阴性短运动杆菌。氧化酶和过氧化氢酶测试的生化反应呈阳性。分析轮廓索引（API 20E）确认为A。

hydrophila isolation with an identity percentage (%id) of 98.4, and the code result was 7,006,127 (Table 1).Table 1 Analytical profile index (API) 20E of isolated A. hydrophila.Full size tableMolecular identification and sequencing analysesThe universal 16 S rRNA gene confirmed the isolation of A. hydrophila.

嗜水气单胞菌分离，同一性百分比（%id）为98.4，编码结果为7006127（表1）。表1分离的嗜水气单胞菌的分析轮廓指数（API）20E。全尺寸表分子鉴定和测序分析通用16 S rRNA基因证实了嗜水气单胞菌的分离。

The obtained sequence of the amplified DNA fragment was blasted on NCBI and registered in GenBank with accession number OR754041.1. Sequence alignment and phylogenetic analyses confirmed the identity of the sequenced isolate, as it clustered in the same clade as other A. hydrophila strains. These sequences exhibited 98.11% similarity with those isolated from stripped catfish and shrimp in Bangladesh (MH220303.1-MH244238.1), 98.11% similarity with those from Procambarus clarkii and estuarine water in India (OP788038.1-MH169204.1), and 97.76% similarity with sequences from crab-cultured water in China (KU570297.1-KU570301.1-KU179356.1) (Fig.

将获得的扩增DNA片段序列在NCBI上进行爆破，并在GenBank中注册，登录号为OR754041.1。序列比对和系统发育分析证实了测序分离株的身份，因为它与其他嗜水气单胞菌菌株聚集在同一进化枝中。这些序列与孟加拉国条纹鲶鱼和虾（MH220303.1-MH244238.1）分离的序列具有98.11%的相似性，与印度克氏原螯虾和河口水（OP788038.1-MH169204.1）的序列具有98.11%的相似性，与中国螃蟹养殖水（KU570297.1-KU570301.1-KU179356.1）的序列具有97.76%的相似性（图）。

2).Fig. 2Phylogenetic tree constructed using neighbors-joining method based on the partial 16S rRNA gene sequence of A.hydrophila.Full size imageIn vitro bactericidal activity of PAA and H2O2 against A.hydrophila.

2） 。图2基于嗜水气单胞菌的部分16S rRNA基因序列，使用邻域连接方法构建的系统发生树。全尺寸图像PAA和H2O2对嗜水气单胞菌的体外杀菌活性。

The results in Table 2 showed that the logarithmic reduction of the average bacterial count of A.hydrophila at 1 mg/L PAA achieved a 5-log reduction after 30 min of contact time, while 20 mg/L H2O2 achieved a 5-log reduction after a contact time of 5 min.Table 2 The mean bactericidal effect (log reduction) of PAA and H2O2 against  A. hydrophila.Full size tableEvaluation of fish sensitivity to PAA and H2O2.

表2中的结果表明，在接触时间30分钟后，1 mg/L PAA下嗜水气单胞菌平均细菌计数的对数减少达到了5 log的减少，而20 mg/L H2O2在接触时间为5分钟后达到了5 log的减少。表2 PAA和H2O2对A的平均杀菌效果（对数减少）。嗜水气单胞菌。全尺寸表评估鱼对PAA和H2O2的敏感性。

Fish in the PAA group (G2) exhibited momentary abnormal swimming behavior, characterized by rapid movement from one side of the aquarium to another and an increase in the escape reflex immediately after adding PAA. These signs rapidly disappeared within 10 min of exposure, and the fish returned to their normal state.

PAA组（G2）中的鱼表现出短暂的异常游泳行为，其特征是从水族馆的一侧快速移动到另一侧，并且在加入PAA后立即增加了逃逸反射。这些迹象在暴露后10分钟内迅速消失，鱼恢复了正常状态。

The H2O2-exposed fish group (G3) did not show any abnormalities in movement or behavior. No mortalities were recorded in either the PAA or H2O2 exposed groups following exposure.Post-exposure challenge and bacterial reisolationThe protective effect of the disinfectants was evaluated by mortality percentage and clinical signs observed in experimentally infected fish in groups G2 and G3.

暴露于H2O2的鱼组（G3）没有显示任何运动或行为异常。暴露后，PAA或H2O2暴露组均未记录死亡率。暴露后攻击和细菌再分离消毒剂的保护作用通过G2和G3组实验感染鱼的死亡率和临床症状来评估。

In the PAA-exposed group (G2), injected fish exhibited 20% mortality, with physical changes including gasping and abnormal swimming behavior but no evident external lesions except for darkening. In contrast, the H2O2-exposed group (G3) showed no mortalities, abnormal behaviors, or clinical signs. Typical A.

在PAA暴露组（G2）中，注射的鱼表现出20%的死亡率，身体变化包括喘息和异常的游泳行为，但除了变黑外没有明显的外部病变。。典型A。

hydrophila was re-isolated and identified from the control positive fish group (G4), which displayed skin darkening, detached scales, extensive petechiae on the base of fins, isthmus, and belly, eroded caudal fins, and eye cataracts. Postmortem examination revealed hemorrhagic and congested gills, liver, kidney, brain, and gall bladder, with mortalities reaching up to 90% (Fig.

。尸检显示鳃、肝、肾、脑和胆囊出血和充血，死亡率高达90%（图）。

3).Fig. 3Control positive experimentally infected fish in fourth group (G4) showed typical signs of A. hydrophila infection in the form of extensive petechiae on base of fins, isthmus and belly with eroded caudal fin (A, B), hemorrhages and congestion in internal organs and brain (C, D).Full size imageHepatic cortisol levelsCompared to the control group, both PAA and H2O2 groups exhibited a signific.

3） 。图3第四组（G4）中对照阳性实验感染的鱼表现出嗜水气单胞菌感染的典型迹象，表现为鳍基部，峡部和腹部广泛瘀斑，尾鳍腐蚀（A，B），内脏和大脑出血和充血（C，D）。全尺寸图像肝脏皮质醇水平与对照组相比，PAA和H2O2组均表现出显着的差异。

Data availability

数据可用性

The data supporting this study’s findings are available from the corresponding author upon request. for the data that is deposited in GenBank: weblink of the accession number (OR754041.1) https://www.ncbi.nlm.nih.gov/nucleotide/OR754041.1?report=genbank&log$=nuclalign&blast_rank=1&RID=8WZ311 × 0013..

支持本研究结果的数据可应要求从通讯作者处获得。对于存放在GenBank中的数据：登录号的网络链接（OR754041.1）https://www.ncbi.nlm.nih.gov/nucleotide/OR754041.1?report=genbank&log$=nuclalign&blast\u等级=1，RID=8WZ311××0013。。

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EPA. Integrated Risk Information System (IRIS). National Center for Environmental Assessment, Office of Research and Development, Washington DC, USA, available on line at (2001). http:/www.epa.gov/iris/.(Download referencesAcknowledgementsThe authors are grateful to Elshaimaa Ismael, Faculty of Veterinary Medicine, Cairo University, for her kind help in the statistical analysis of data.FundingNot applicable.Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).Author informationAuthors and AffiliationsDepartment of Veterinary Hygiene and Management Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptAbdelrhman Gamal, Asmaa Metwally Ali & Hanan S.

美国环保局。。美国华盛顿特区研究与发展办公室国家环境评估中心，可在线获取（2001）。http://www.epa.gov/iris/。（下载参考文献致谢作者感谢开罗大学兽医学院Elshaima Ismael在数据统计分析方面的善意帮助。资助不适用。科学，技术与创新资助局（STDF）与埃及知识银行（EKB）合作提供的开放获取资金。作者信息作者和附属机构开罗大学兽医卫生与管理学院兽医学系，吉萨，12211，EgyptAbdelrhman Gamal，Asmaa Metwally Ali＆Hanan S。

KhalefaDepartment of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptDalia A. Abdel-moneamDepartment of Physiology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptAsmaa Safwat MorsiDepartment of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptNermeen M.

开罗大学兽医学院水生动物医学与管理系，吉萨，12211，埃及达利亚A.阿卜杜勒·莫奈姆生理学系，开罗大学兽医学院，吉萨，12211，埃及阿斯玛·萨夫瓦特·莫尔斯食品卫生与控制系，开罗大学兽医学院，吉萨，12211，埃及内梅恩M。

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PubMed Google ScholarContributionsA.G., D.A.A and H.S.K.: Conceptualization, methodology, formal analysis, data curation, writing–original draft, writing–review and editing. N.M., A.A.M., and A.S.M.: Methodology, formal analysis, data curation, writing original draft. All authors have read and agreed to the submitted version of the manuscript.Corresponding authorsCorrespondence to.

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The study protocol was reviewed and approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt (Vet CU 25122023842). Our study is reported following ARRIVE guidelines.

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Reprints and permissionsAbout this articleCite this articleGamal, A., Abdel-moneam, D.A., Morsi, A.S. et al. In-vitro and in-vivo assessment of the bactericidal potential of peracetic acid and hydrogen peroxide disinfectants against A. hydrophila infection in Nile tilapia and their effect on water quality indices and fish stress biomarkers..

转载和许可本文引用本文Gamal，A.，Abdel moneam，D.A.，Morsi，A.S.等人体外和体内评估过氧乙酸和过氧化氢消毒剂对尼罗罗非鱼嗜水气单胞菌感染的杀菌潜力及其对水质指数和鱼类应激生物标志物的影响。。

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KeywordsPeracetic acidHydrogen peroxide

关键词消旋酸过氧化氢

A. hydrophila

A、 嗜水气单胞菌

Bacterial countCortisolAntioxidant capacity

细菌计数皮质醇抗氧化能力