Characterization of Escherichia coli O86 O-antigen gene cluster and identification of O86-specific genes

Escherichia coli O86 belongs to the enteropathogenic E. coli (EPEC) group, some strains of which are pathogens of humans, wild birds and farm animals. The O-antigen gene cluster of E. coli O86 was amplified by long-range PCR using primers based on the housekeeping genes galF and gnd, and then sequenced. Genes involved in GDP-Fuc and N-acetyl-galactosamine (GalNAc) synthesis and genes encoding glycosyltransferases, O-unit flippase and O-antigen polymerase were identified on the basis of homology. By screening against 186 E. coli and Shigella-type strains, two genes specific to E. coli O86 were identified. A polymerase chain reaction (PCR) assay, based on the specific O-antigen genes identified here, could be used for the rapid detection of E. coli O86 in environmental and clinical samples. The relationship between E. coli O86 and O127 was also determined by comparing the two O-antigen gene clusters.     

Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces

Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Non-O157 STEC, particularly O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. Our objective was to develop a multiplex PCR to distinguish O157 and the 'top six' non-O157 serogroups (O26, O45, O103, O111, O121, and O145) and evaluate the applicability of the multiplex PCR to detect the seven serogroups of E. coli in cattle feces. Published sequences of O-specific antigen coding genes, rfbE (O157) and wzx and wbqE-F (non-O157), were analyzed to design serogroup-specific primers. The specificity of amplifications was confirmed with 138 known STEC strains and the reaction yielded the expected amplicons for each serogroup. In feces spiked with pooled 7 STEC strains, the sensitivity of the detection was 4.1 × 10(5)CFU/g before enrichment and 2.3 × 10(2) after 6h enrichment in E. coli broth. Additionally, 216 fecal samples from cattle were collected and tested by multiplex PCR and cultural methods. The multiplex PCR revealed a high prevalence of all seven serogroups (178 [O26], 108 [O45], 149 [O103], 30 [O111], 103 [O121], 5 [O145], and 160 [O157]) of 216 samples in fecal samples. Cultural procedures identified 33.1% (53/160) and 35.5% (11/31) of PCR-positive samples for E. coli O157 and non-O157 serogroups, respectively. Samples that were culture-positive were all positive by the multiplex PCR. The multiplex PCR can be used to identify serogroups of putative STEC isolates.     

Escherichia coli strains from edema disease: O serogroups, and genes for Shiga toxin, enterotoxins, and F18 fimbriae

The objectives of the research were to determine the presence of the gene sequences for Shiga Toxin 2e (Stx2e), enterotoxins (ST-I, ST-II and LT-I), and F18 fimbriae in 144 Escherichia coli strains isolated from pigs with edema disease; to assess the ability of stx2e(+) strains to produce Stx2e; and to determine the O serogroups of the E. coli strains. Presence of the genes was determined by polymerase chain reaction (PCR), production of Stx2e was assessed by cytotoxicity for Vero and Hela cells, O serogroups were identified by agglutination with specific antisera. Of the 144 strains tested, 99 were stx2e(+) by PCR, but only 45 of these were Stx2e(+) in the cell culture assays. Among the 99 stx2e(+) strains, PCR detected the genes for F18ab, ST-I, ST-II, LT-I in 76, 40, 31 and 16 strains, respectively. Forty-one of the 99 sxt2e(+) strains belonged to O group 139; the rest did not belong to the classical edema disease O serogroups. It is likely that the enterotoxins, whose genes were detected at high frequency, are responsible for diarrhea seem in pigs with edema disease in Brazil.     

Characteristics of verotoxigenic Escherichia coli from pigs.

Porcine verotoxigenic Escherichia coli were characterized with respect to frequency of occurrence, serogroup, and association with disease, weaning, and selected properties of the bacterium. Of 668 strains of E. coli from southern Ontario pigs with enteric disease, 32 (4.8%) produced verotoxin at 10(3)-10(7) cytotoxic doses per mL of culture supernatant. Of 22 isolates which belonged to O serogroups 138, 139 and 141, 15 produced verotoxin. Among other enterotoxigenic types of E. coli, two of 57 isolates of O157:K"V17" and two of 96 isolates of O149:K91 were verotoxigenic. The remaining 13 verotoxigenic E. coli belonged to O groups 2, 107, 120, 121 and 130. An additional 21 verotoxigenic E. coli belonging to O groups 138, 139 and 141 and three to O157:K"V17" were identified in a collection of 47 E. coli recovered from weaned pigs with enteric disease. Verotoxigenic E. coli were associated with postweaning diarrhea, bloody stools, sudden death and edema disease. They were isolated at similar frequencies (14%) from healthy weaned pigs, and from weaned pigs with enteric disease. Isolation rates from neonates were low and significantly different from rates in weaned pigs. Neutralizing antibody to verotoxin was not detected in the sera of 45 pigs, which included pigs from herds with a history of edema disease. Verotoxin was not associated with production of colicin, hemolysin, or enterotoxins or with any of 23 biochemical properties of the organisms. The serological data indicate that porcine verotoxigenic E. coli are not a common source of verotoxigenic E. coli for humans. Porcine verotoxin may play a role in postweaning diarrhea and absence of detectable neutralizing antibody in serum may be an important aspect of pathogenesis.     

Phenotyping of E. coli serotypes associated to oedema disease

Background

Oedema disease is a severe disease, mainly affecting recently weaned pigs. It is caused by E. coli strains that express fimbriae F18 and produce verotoxin 2e, mainly belonging to serotype O138, O139 or O141. The aim of this study was to compare E. coli isolates within these serotypes with respect to diversity.

Methods

Faecal E. coli strains belonging to serotypes O138, O139 and O141 isolated during the period 1994–1998 from Swedish pigs aged less than 12 weeks were compared using a biochemical fingerprinting system. Aiming to compare the results obtained over time, also strains isolated during 1964–67 and 1975–80 were included in the study. The study comprised 129, 263 and 95 isolates of E. coli serotype O138, O139 and O141, respectively.

Results

Biochemical phenotypes (BPTs) were defined. At each sampling occasion each herd could only contribute with one isolate per BPT. Consequently, all but one of identical BPTs identified at a specific sampling occasion was omitted. The final number of isolates from 1994–98 that was compared included 64, 182 and 41 isolates of serotypes O138, O139 and O141, respectively. Within each serotype, the dominating BPT included over 65% of the compared isolates, demonstrating a large dominance of one BPT per serotype. These dominating BPTs were also demonstrated in the material from the 1960ies and the 1970ies. Still, the presence of other common BPTs (especially within serotype O138 and O139) demonstrated a certain variation within serotype. In a herd severely affected by oedema disease, E. coli serotype O139 was easily demonstrated in diseased pigs but only rarely in apparently healthy weaners

Conclusion

The results obtained demonstrate the presence of dominating BPTs within the oedema disease inducing serotypes. A stability of these BPTs over time was observed, presumably at least partly due to a never-ending access to naïve pigs. Still, the presence of other common BPTs indicates a variation over time, which visualises the importance of monitoring for this. Such studies should focus on pigs affected by oedema disease, because oedema disease inducing strains of E. coli were only rarely demonstrated in healthy pigs in a herd affected by oedema disease.     

Prevalence and deletion types of the pathogenicity island ETT2 among Escherichia coli strains from oedema disease and colibacillosis in pigs

Piglet pathogenic Escherichia coli encoding Shigatoxin 2e and F18 adhesins are the etiological agents of oedema disease as well as of non-oedema disease colibacillosis. In order to reveal virulence differences among this pathogen, the presence of the pathogenicity island (PAI) E. coli type three secretion system 2 (ETT2) was examined. Using PCR and Southern blot techniques for the identification of the right, the middle, and the left region of this 29.9kb large genetic element, the entire ETT2 was found among E. coli O138:H(-), O139:H1, and O147:H6 strains originated from cases of oedema disease in Germany between 1995 and 2001 and belonging to various clonal types. In contrast, non-oedema disease E. coli isolates (e.g. O8:H19, 101:H(-), O141:H4) contain deleted subtypes of ETT2. These deletions cover the translocon part of the putative ETT2-encoded type III secretion apparatus. It is suggested that the entire ETT2 is associated with a particular virulence trait of piglet oedema disease E. coli (EDEC).     

海安县猪水肿病的流行病学与病原特性研究

对江苏省海安县2003年5月～2004年5月各乡镇猪水肿病的发病和死亡情况进行了流行病学调查，发现猪水肿病在海安地区呈零星散发性发生，发病率为0.31％～1．84％，死亡率为0．07％—0．56％，病死率为16．0％～35．0％。水肿病主要侵害仔猪，育成猪也偶有发生，一般以气温比较高的季节多发。同时从疑似猪水肿病的病料中分离到12株大肠杆菌，经O血清型鉴定，5株为0139，2株为O45，O55、O93，O9、O101、O119各1株。经多重PCR检测毒素基因(STa，STb、LT、SLT-2e)和大肠杆菌粘附素单抗(F4、F5、F6、F41、F18)检测茵毛，共6个分离株带有SLT-2e基因，其中O139分离株5株，055分离株1株，其余6株均带STb基因。12个分离株中，单独表达F18菌毛的4株(O1392株，O45、O119各1株)，F5 F411株(O93)，F41 F181株(O139)，F5 F41 F182株(均为O139)。经药敏试验，多数分离株对壮观霉素和新霉素高度敏感。     

Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea

Duplex real-time PCR assays were used as modules to cover partially automated detection of 12 genes encoding adhesins, enterotoxins and Shiga toxins in faecal E. coli isolates. For this a total of 194 E. coli isolates from pigs suffering from post-weaning diarrhoea (PWD), including 65 isolates with haemolytic activity, and 83 isolates from calves with diarrhoea were examined. Data obtained by PCR were compared with O-typing and with haemolytic activity as indirect virulence markers. E. coli O-types O139:K82, O141:K85, and O149:K91 accounted for 43.8% (n = 85) of all porcine strains and for 55.4% (n = 36) of the porcine strains, which exhibited haemolytic activity. These strains carried virulence genes by 65.9% (n = 56) and 80.6% (haemolytic E. coli, n = 29), respectively. The E. coli O-types O139:K82 and O141:K85 were significantly associated with the adhesin gene F18, and O149:K81 with the F4 gene. In this context, detection of the gene encoding F18 was coupled predominantly with the genes responsible for the production of the toxins ST-I, ST-II and Stx2, and the F4 gene with those of the enterotoxins ST-I, ST-II and LT. Both virulence patterns were detected more pronounced in E. coli strains with haemolytic activity. Fifty-six of a total of 83 E. coli isolates originating from calves were O-typed as O101 (O101:K28, O101:K30, O101:K32; n = 29), O78:K80 (n = 23), and O9:K35 (n = 4). Most of the E. coli O78:K80 strains carried the F17 gene (69.6%, n = 16). Virulence genes encoding for F4, F5 or ST-I were detected only in single cases. Intimin and Shiga toxin genes that are present in enterohaemorrhagic E. coli (EHEC) were not detected.     

动物性食品源大肠杆菌O血清型鉴定及其K88菌毛基因检测

本研究对河北省冀东地区农贸市场和超市采集的生猪肉、生鸡蛋和生羊肉等分离得到的20株大肠杆菌进行大肠杆菌血清型鉴定;并检测不同血清型大肠杆菌的K88菌毛基因。采用常规方法进行大肠杆菌的O血清型鉴定,用PCR方法检测K88菌毛基因。分离鉴定的20株大肠杆菌有7种血清型,包括O38、O78、O88、O11、O107、O91、O9,其中O38、O78为优势血清型菌,均占分离菌株的25%(5/20)。在分离的动物性食品源大肠杆菌中有30%(6/20)的菌株K88菌毛基因扩增呈阳性。结果表明,O78、O38为冀东地区动物性食品源大肠杆菌常见血清型,30%(6/20)的菌株K88菌毛基因扩增阳性。     

Detection of O antigens in Escherichia coli

Lipopolysaccharide on the surface of Escherichia coli constitutes the O antigens which are important virulence factors that are targets of both the innate and adaptive immune systems and play a major role in host-pathogen interactions. O antigens are responsible for antigenic specificity of the strain and determine the O serogroup. The designation of O serogroups is important for classifying E. coli strains, for epidemiological studies, in tracing the source of outbreaks of gastrointestinal or other illness, and for linking the source to the infection. For conventional serogroup identification, serotyping by agglutination reactions against antisera developed for each of the O serogroups has been used. In the last decade, many O-antigen gene clusters that encode for the enzymes responsible for the synthesis of the variable oligosaccharide region on the surface of the bacteria have been sequenced and characterized. Unique gene sequences within the O-antigen gene clusters have been targeted for identification and detection of many O groups using the polymerase chain reaction and microarrays. This review summarizes current knowledge on the DNA sequences of the O-antigen gene clusters, genetic-based methods for O-group determination and detection of pathogenic E. coli based on O-antigen and virulence gene detection, and provides perspectives on future developments in the field.     

Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.     

Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.     

仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌（EPEC）、肠产毒性大肠杆菌（ETEC）和肠聚集性大肠杆菌（EAEC）,毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

Isolation,Identification and Drug Resistance Analysis of Interstinal Pathogenic Escherichia coli and Salmonella Isolated from Diarrhea Piglets

In order to find out the serotype, resistant phenotype and genotype of Escherichia coli (E. coli) and Salmonella in piglets, this study collected 128 samples of diarrhea piglets from seven large-scale pig farms in five cities in Guizhou province, and the E. coli and Salmonella were isolated and identified. The pathogenicity of the strain was identified by animal test. The drug resistance of the main pathogen was tested by drug susceptibility paper. The resistance gene of each pathogen was detected by PCR. The drug resistance and genotype correlation of the bacterial were analyzed. The results showed that 78 strains of pathogenic E. coli and 21 strains of Salmonella were isolated and identified in this study. The serotypes of pathogenic E. coli were predominantly O138 and O87. Salmonella Typhimurium and Salmonella Enteritidis were predominant serotypes. The susceptibility test showed that the resistant strains of 78 strains of E. coli were more than 80% resistant to β-lactams and more than 40% for other antibacterials. The resistance rate of 21 strains of Salmonella to aminoglycosides was more than 50% and more than 20% to other types of antibacterials; 12 and 10 kinds of drug resistance-related genes of E. coli and Salmonella were detected, respectively; The coincidence rate of resistant genotype and phenotype of two kinds of bacteria were above 60%, and both were multiple drug resistance. This study provided a theoretical basis for comprehensive prevention and control of piglets diarrhea.     

腹泻仔猪肠道致病性大肠埃希氏菌和沙门氏菌的分离鉴定与耐药性研究

为探明仔猪细菌性腹泻肠道致病性大肠埃希氏菌和沙门氏菌流行的血清型、耐药表型及耐药基因型,本试验采集了贵州省5个地（州）市7个规模化养猪场的128份腹泻仔猪肠道样本,并对采集的样本进行了大肠埃希氏菌和沙门氏菌分离与鉴定,通过动物试验鉴定菌株的致病性,利用血清学方法鉴定其血清型,并通过药敏纸片法对主要致病菌进行耐药性研究,采用PCR技术检测各致病菌株耐药相关基因,分析细菌耐药表型和耐药基因型相关性。结果显示,本研究共分离鉴定到78株致病性大肠埃希氏菌与21株沙门氏菌,致病性大肠埃希氏菌血清型以O138、O87为主,沙门氏菌血清型以鼠伤寒沙门氏菌、肠炎沙门氏菌居多;药敏试验结果表明,本试验分离到的78株致病性大肠埃希氏菌对β-内酰胺类药物耐药率达80%以上,对其他种类的抗菌药耐药率均超过40%,分离鉴定的21株沙门氏菌对氨基糖苷类药物耐药率达50%以上,对其他种类的抗菌药耐药率均达20%以上;本试验分离鉴定的致病性大肠埃希氏菌共检出12种耐药相关基因,沙门氏菌共检出10种耐药相关基因,两种细菌耐药基因型与耐药表型符合率均达60%以上,且均为多重耐药。本研究为仔猪腹泻的综合防控提供了理论依据。     

Identification of Escherichia coli O157:H7-strains from pigs with postweaning diarrhoea and amplification of their virulence marker genes by PCR

Escherichia coli isolated from pigs with postweaning diarrhoea were examined by PCR for the presence of the O157 rfb gene responsible for the biosynthesis of E coli O157 lipopolysaccharide. Among the 372 isolates tested, 38 (10.2 per cent) were of the O157 serogroup, but none of these possessed the H7 determinant. Further analysis of the E coli O157 isolates revealed that seven of them had the genes responsible for the production of Shiga toxin 1 and eaeA intimin, four other strains had genes responsible for the production of Shiga toxin 2, and four other strains were positive for the enterohaemolysin gene.     

青海省部分仔猪水肿病病原的血清型鉴定

就青海省仔猪水肿病多发区分离鉴定的45株溶血性大肠杆菌进行了血清学定型，结果表明：已鉴定出血清型的菌株33株，占73．3％，12株未定出血清型，占26．7％；其中O8 10株(22．2％)，O25 1株(2．2％)，O101 1株(2．2％)，O138 3株(6．7％)，O139 7株(15．5％)，O141 5株(11．1％)，O149 6株(13．3％)。O抗原优势菌株分别为：O8、O139、O141、O139，占全部菌数的62．2％。     

Characterization of Escherichia coli strains isolated from pigs with edema disease.

Escherichia coli strains belonging to serogroups O 138 and O 139 isolated from pigs with edema disease, were characterized with respect to the presence of genes encoding Shiga-like toxin I, Shiga-like toxin II and Shiga-like toxin IIv (SLT I, SLT II and SLT IIv). Genes coding for the heat-stable and heat-labile enterotoxins (ST I and LT I) were also detected. Plasmid profiling, restriction enzyme digestion of total DNA, and ribotyping were performed for further characterization of the strains. The oligonucleotide probes applied in this study appeared to be useful tools for detecting genes coding cytotoxins and enterotoxins. DNA from 12 of 16 strains hybridized with two SLT II probes, and DNA from two SLT IIv encoding strains also hybridized with the ST I probe. DNA from one SLT IIv negative strain hybridized with the LT I probe. The results from plasmid profiling, restriction enzyme digestion, and ribotyping were compared with serogrouping in attempts to distinguish between the different E. coli edema disease isolates. Fourteen different plasmid profiles were identified, and as restriction patterns barely did, and ribotyping patterns did not, reveal any information useful for differentiation of the strains beyond serogroup level, plasmid profiling seemed to be the most suitable method for discrimination between the edema disease strains investigated here.     

Surveillance of dairy production holdings supplying raw milk to the farmhouse cheese sector for Escherichia coli O157, O26 and O111

Clinically healthy domestic animals can harbour Escherichia coli O157 and other verocytotoxigenic E. coli (VTEC) strains in their faeces. Milk filters can be used to microbiologically monitor direct milk secretion and environmental contamination for these pathogens. The aim of this study was to establish baseline data on the prevalence and characteristics of VTEC organisms in lactating animals (bovine, ovine and caprine) supplying milk to the farmhouse cheese sector, with particular emphasis on serogroups O157, O111 and O26. Fifty-six bovine, 13 caprine and 5 ovine herds/flocks, the majority of which supplying milk for farmhouse cheese production, were surveyed from May 2004 to July 2005. Milk filters were analysed by immunomagnetic separation followed by PCR, on a serogroup-specific basis for E. coli O157, O26 and O111. Positive isolates were examined using a multiplex PCR protocol, for their potential to produce verocytotoxins (vt1/vt2), the haemolysin-encoding gene (hlyA) and the gene encoding attaching and effacement (eae). Five verocytotoxigenic and 22 non-virulent E. coli O157 isolates were detected. Seventeen E. coli O26 isolates were also detected, four of which were verocytotoxigenic, seven isolates contained the eae gene only and six isolates were devoid of any of the virulence factors. The VTEC O157 and O26 isolates contained the hlyA and eae genes along with the verocytotoxin genes. No E. coli O111 isolates were detected. Some of the herds were positive on more than one occasion and multiple E. coli serogroups were isolated from the same milk filter sample. Although all food products tested were VTEC negative, routine surveillance for such pathogens in raw milk/raw milk products is of public health importance. Herd-level surveillance along with subsequent risk management action may be a cost-effective component of risk reduction strategies for food production, drinking water supplies and the protection of public health.     

北京鸭传染性窦炎的调查研究Ⅱ、病原学与人工感染试验

本实验从40只患传染性窦炎的病鸭眶下窦中(其中4只未作病毒分离)分离到16株血凝性病毒,经血凝抑制试验(HI)及神经氨酸酶抑制试验(NI)鉴定,均为A型流感病毒(H_(11)-N_9);又分离到18株霉形体中,除2株未定种外,其余16株经生长抑制试验和间接表面免疫荧光试验鉴定均为鸭霉形体(Mycoplasma anatis)。同时分离到31株大肠杆菌,O抗原分属O_(15)、O_(70)、O_(138)三个血清型。将A型流感病毒(H_(11)N_9)、鸭霉形体和大肠杆菌(O_((?))和O_(138))分别经眶下窦接种1日龄雏鸭,并接种灭菌生理盐水作对照。结果,鸭霉形体的感染成功地复制出自然病例,表明鸭霉形体是鸭传染性窦炎的病原体。