普氏蹄蝠胃肠道菌群的多样性

【目的】研究普氏蹄蝠(Hipposideros pratti)胃肠道菌群多样性及致病菌的种类。【方法】采用Mi Seq高通量测序技术,通过对16S r RNA基因V1-V2区基因进行测序,研究普氏蹄蝠胃肠道细菌的群落组成。应用MG-RAST V3.3.6分析和统计样品序列和操作分类单元(OTU)数目,分析胃肠道菌群物种丰度,并进行聚类分析。【结果】从普氏蹄蝠胃和肠道中分别获得144 998条和275 274条原始序列以及48 332条和91 758条有效序列,分属于894个和756个操作分类单元。胃中菌群丰度指数Chao指数(1 490)和ACE指数(2 221)分别低于肠道菌群的Chao指数(2 051)和ACE指数(3 556);Shannon多样性指数(2.405)低于肠道(2.407);Simpson多样性指数(0.168)高于肠道(0.151)。系统进化分析表明胃肠中的细菌主要分布在6个门,均以变形菌门(Proteobacteria)(胃中占56.4%,肠中占46.0%)和厚壁菌门(Firmicutes)(胃中占40.7%,肠中占49.2%)为优势菌门。胃肠道中丰度在0.1%以上的属有24个,胃中优势类群为乳球菌属(Lactococcus)和哈夫尼菌属(Hafnia),分别占整个菌群的26.1%和21.0%;肠道中优势类群为肠球菌属(Enterococcus)和沙门氏菌属(Salmonella),分别占整个菌群的15.2%和12.7%。普氏蹄蝠胃肠道中的优势菌群均为人类的致病菌或者条件致病菌。【结论】普氏蹄蝠携带有大量人类致病菌。因此,应注意防止向人类传播。     

南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

团水虱不同生长阶段肠道微生物多样性分析

为了解团水虱不同生长阶段的肠道微生物多样性,采用Mi Seq高通量测序技术和生物信息学分析手段,构建团水虱幼体、稚体和成体三个生长阶段肠道微生物的16S r RNA基因测序文库,分析三个样品中的菌群组成和生物多样性。结果表明,成体(T1)、稚体(T2)、幼体(T3)三个生长阶段肠道微生物样品共产生794个OTU,分别含646个、686个和156个OTU。分析显示,共检测到25个细菌门类,其中包括变形菌门、拟杆菌门、梭杆菌门等3个优势菌门。变形菌门为三个样品的共同优势菌,在幼体、稚体和成体中分别占51.7%、55.1%和51.6%;拟杆菌门为稚体(30.21%)和成体(38.57%)的次优势菌门,在幼体肠道内低于5%;梭杆菌门在幼体体内占37.8%,是幼体的次优势菌门,但在稚体和成体体内均低于2%。幼体与其他两个生长阶段的肠道细菌群落结构差异显著,与稚体和成体的共有OTU数量,占检测到的OTU总数的比例均低于25%。其中稚体肠道细菌有着最高丰富度及多样性,其Chao指数为697.15、Shannon指数为4.69,最低的Simpson优势度指数为0.035 1,而幼体肠道细菌丰富度和多样性最低。研究结果表明,团水虱肠道菌群种类丰富,幼体肠道菌群种类及其多样性与稚体、成体差异明显;菌群变化与各生长阶段团水虱生活习性关系密切。     

基于16S rRNA基因序列分析1例健康人龈上菌群

目的构建细菌16S rRNA基因文库分析健康人龈上菌群的组成。方法取1例健康成年女性龈上菌斑并构建细菌16S rRNA基因文库,分析其龈上菌群组成。结果 1例健康人龈上菌斑细菌的种水平分类有62种,其中可以培养的细菌有34种而尚无法培养的细菌有28种(45.1%);新发现的细菌物种有17种(27.4%);缓症链球菌是克隆子数最多的优势菌种;链球菌属、奈氏菌属和嗜血杆菌属占文库的60%,为主要菌群;构建的细菌16S rRNA基因文库的覆盖率为95%,文库的均匀度值为0.016。结论 1例健康人龈上菌群中45.1%的细菌尚无法分离培养、27.4%的物种尚不清楚,未培养菌及尚不清楚的菌种中可能藏匿着与口腔疾病密切相关的致病菌,全面地了解健康人龈上菌群的组成有助于研究龋病的发病机制。     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

柠檬酸废水IC反应器厌氧颗粒污泥真细菌结构分析

目的:分析柠檬酸工业废水IC厌氧反应器处理时产生的厌氧颗粒污泥中真细菌的菌群结构.方法:构建细菌的16S rDNA克隆文库,对文库中的16S rDNA基因序列进行测序,然后Blast比对,并进行分类、建系统发育树.结果:对获得的77个16S rDNA序列进行测序,按序列相似性≥97％的分类标准,这些序列可分为22个OTU,其中4个优势OTU分别与棒杆菌属(Corynebacterium)、梭菌属(Clostridium)、消化球菌属(Peptococcus)、疣微菌属(Verrucomicrobia)最为相近,其余OTU的克隆数较少.颗粒污泥中的真细菌主要为放线菌纲(Actinobacteria)、梭菌纲(Clostridia)、拟杆菌纲(Bacteroidetes)以及δ-变形菌纲(Deltaproteobacteria)的细菌,分别占克隆总数的34/77、31/77、6/77、6/77.结论:该文研究了柠檬酸废水处理过程中产生的厌氧颗粒污泥中细菌的菌群组成和结构,为深入了解柠檬酸废水的厌氧处理过程提供了一定的理论借鉴作用.     

石棉地区藏族、彝族和汉族人群肠道微生物菌群构成与分布

目的 探讨石棉地区藏族、彝族和汉族人群肠道微生物菌群构成分布情况。 方法 将2017年7月至2019年7月在我院进行健康体检的并符合纳入和排除标准的125例健康人作为研究对象,其中汉族65例,彝族32例,藏族28例。收集受试健康人群清晨空腹粪便标本,利用高通量测序技术测定石棉地区藏族、彝族和汉族人群粪便样本16S rRNA序列并建立分类操作单元(OTU),分析肠道微生物结构特征及优势菌群,并对各民族人群肠道微生物进行聚类分析。 结果 共获得514 069条高质量16S rRNA序列,每个样品平均生成序列数为(4 112.55±1 258.67)条;97%相似度归并共获得68 232个OTU,每个样品平均OTU为(545.86±157.49)个;石棉地区汉族人群粪便标本中占优势细菌门为拟杆菌门和厚壁菌门,彝族及藏族人群粪便标本占优势细菌门为厚壁菌门和拟杆菌门,菌门水平上丰富度差异存在统计学意义(F=13.810,P48%。 结论 石棉地区藏族和彝族与汉族人群间肠道微生物优势细菌门及细菌属虽相似但在丰富度上存在差异,汉族健康人群肠道微生物菌群呈现不规律、分散聚类现象,彝族、藏族健康人群肠道微生物菌群呈现集中聚类现象,临床应根据种族差异对人群进行膳食指导以调控肠道微生态平衡。     

脊尾白虾肠道微生物菌群结构

【目的】研究海水池塘养殖条件下的脊尾白虾肠道微生物菌群组成及多样性。【方法】采用PCR-RFLP技术,以直接提取的脊尾白虾肠道细菌总DNA为模板进行16S rRNA 基因扩增,产物与T载体连接建立质粒文库,从RFLP建立的文库中筛选出不同细菌来源的克隆子,将测定的差异克隆子16S rRNA片段序列与GenBank数据库进行比对。【结果】从脊尾白虾肠道的菌群文库中共获得114个克隆子,Hae Ⅲ和Msp I双酶切得到11种差异克隆子;对差异克隆子测序后,其中8个差异序列与已知细菌具有较高的同源性,分别是假单胞菌属(Pseudomonas)、肠杆菌属(Enterobater)、冰冻小杆菌属(Frigoribacterium)、褐杆菌属(Phaeobacter)、弧菌属(Vibrio)、赤杆菌属(Erythrobacter)、气单胞菌属(Aeromonas)、葡萄球菌属(Staphylococcus)和其他未培养细菌。【结论】初步揭示了脊尾白虾肠道微生物菌群结构的组成,为开发脊尾白虾专用微生态制剂提供基础资料。     

210株肠道致病菌的分类及耐药性分析

目的：探讨本地区肠道致病菌的分类及主要致病菌的耐药性,给临床治疗腹泻疾病提供指导。方法：药敏试验采用K-B法,用WHONET4软件进行数据分析。结果：210株肠道致病菌中,志贺菌属占70％,沙门菌属占3％,致病性大肠埃希菌占4．8％,副溶血弧菌占3．8％,阴沟肠杆菌占3．3％,鼠伤寒沙门菌占2．4％,霍乱弧菌占1．9％,其他占10．8％等;在志贺菌属中,福氏占87％,宋内占13％。福氏和宋内志贺菌对三代头孢和IMP较敏感,对复方新诺明耐药率较高。结论：福氏志贺菌是肠道的主要致病菌;临床怀疑菌痢时,应首选三代头孢、IMP。     

斑头雁成鸟与雏鸟泄殖腔微生物的对比分析

肠道微生物通过维持稳态、辅助消化和促进免疫系统发育等方式维护宿主的健康状态。肠道微生物本身则受到宿主的基因、饮食、年龄和环境等因素的影响。然而,肠道微生物的变化与宿主年龄之间的关系仍有许多未知。本研究分别收集斑头雁(Anser indicus)2只成鸟及3只雏鸟泄殖腔样品,提取肠道微生物总DNA,采用16S rRNA高通量测序的方法,分析并比较两年龄阶段鸟类肠道微生物的菌群结构及组成差异。研究发现,斑头雁雏鸟泄殖腔微生物属于9个门,含量最高的前5个门分别是梭杆菌门(48.29%)、厚壁菌门(22.21%)、变形杆菌门(22.07%)、放线菌门(5.02%)和软壁菌门(1.93%)。成鸟泄殖腔微生物属于17个门,最多的依次是变形菌门(64.69%)、厚壁菌门(23.92%)、蓝细菌(8.48%)、放线菌门(1.43%)和梭杆菌门(0.56%)。在属的水平,斑头雁雏鸟泄殖腔微生物属于18个属,而成鸟含有24个属。成鸟泄殖腔微生物的α多样性显著高于雏鸟(P0.05,Welch′s t-test)。有186个操作分类单元(OTU)属于成鸟和雏鸟共有,而其他640个OTU和90个OTU则分别隶属于成鸟和雏鸟。雏鸟中67.39%的OTUs是成鸟所具有的。基于OTU的聚类结果与年龄分组一致。本结果对认识鸟类肠道微生物与宿主年龄变化之间的关系有一定的参考价值。     

Determination of Vibrio scophthalmi and its phenotypic diversity in turbot larvae

The association of Vibrio scophthalmi with turbot larvae was assessed, by molecular methods with a species-specific probe, in the rearing stages of turbot (Scophthalmus maximus) larvae using a routine batch of production at a fish farm. The phenotypic diversity of this bacterial species was also studied to identify predominant phenotypes at successive stages of larval development. Vibrio scophthalmi was detected in all turbot larvae samples except in the sample from day 0 after hatching. The percentage of V. scophthalmi in the intestinal microbiota increased throughout larval development. Vibrio scophthalmi was also detected in live food (brine shrimps) and water from the tanks, but not in the sediment. All turbot larvae, 15-57 day old, showed several V. scophthalmi phenotypes, and a pattern of successive waves of phenotypes was observed during successive larval stages. This indicates that certain strains may colonize the intestine more efficiently and thus maintain their population for longer than other strains. Vibrio scophthalmi populations from turbots of different origin were very similar, suggesting that irrespective of geographical area, turbot populations share similar V. scophthalmi strains. Vibrio scophthalmi strain was not isolated from other cultured fish, only turbot larvae, at the same hatchery receiving water from the same supply.     

养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析

2004年夏季山东海阳一带养殖大菱鲆(Scophthalmus maximusL.)暴发溃疡病。症状主要表现为体表病灶部位出血、肌肉溃烂、眼球凸出等,解剖可见鳃丝贫血,肝脏充血,肾脏和胆囊肿大,肠壁充血并呈透明状,从出现发病症状起,大约一周后开始出现死亡。从病鱼体表溃疡部位及内脏分离出优势菌并命名为H1,经人工感染证实H1即为引起本次养殖大菱鲆体表溃疡症的病原菌。对病原菌进行鉴定揭示该菌革兰氏染色阴性,菌体呈短杆状,极生单鞭毛。综合该菌在形态、生理生化、API20NE与API20E自动鉴定结果、16S rDNA同源性等方面的特性,确认H1为哈维氏弧菌(Vibrio harveyi),该菌对呋喃妥因、菌必治等多种抗生素敏感。哈维氏弧菌是海水养殖鱼类的常见致病菌,但作为养殖大菱鲆的病原菌属首次报道。     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin

AIMS: The aim of the present study was to clarify the taxonomic status of intestinal bacteria isolated from Japanese flounder (Paralichthys olivaceus) and describe their ability to digest chitin. METHODS AND RESULTS: Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences showed that 82 representative isolates were closely related to three major species of marine vibrios, Vibrio scophthalmi-Vibrio ichthyoenteri group (41 isolates), Vibrio fischeri (39 isolates) and Vibrio harveyi (two isolates), with similarities of 97.2-99.8%, 96.4-100% and 98.6-99.5% respectively. These findings indicate that V. scophthalmi-V. ichthyoenteri group is indigenous to the intestinal tract of Japanese flounder. Moreover, the ability of 82 isolates to digest chitin was examined using the agar plate method and PCR amplification of the chiA gene. The two V. harveyi isolates and 36 of 41 V. scophthalmi-V. ichthyoenteri isolates digested chitin and were chiA PCR positive, whereas all 39 V. fischeri isolates digested chitin but were chiA PCR negative. CONCLUSIONS: Intestinal bacteria from Japanese flounder were mainly composed of Vibrio scophthalmi-V. ichthyoenteri group and V. fischeri. Taken together, the results showed that 81 of 82 isolates could digest chitin. However, only 38 of these isolates possessed a chiA homologue which could be identified by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that Japanese flounder harbours bacteria of the V. scophthalmi-V. ichthyoenteri group, and these results are similar to what has been found for turbot (Scophthalmus maximus).     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.     

Non-isotopic detection of Tetramicra brevifilum (Microspora) DNA in turbot tissues.

A non-isotopic in situ hybridization (ISH) method was developed for detection of Tetramicra brevifilum, a commercially important parasite in farmed turbot Scophthalmus maximus. The probe relies on sequences from the small-subunit rRNA gene (SSUrDNA) of Tetramicra brevifilum and was obtained by polymerase chain reaction then labeled with digoxigenin. The results obtained demonstrate that the probe hybridizes well with genomic DNA of the spores; thus, it is an effective method for detecting multiorgan infections of turbot by T. brevifilum.     

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.