Biochemical typing of Actinobacillus pleuropneumoniae

A study was conducted to evaluate the possibility of using biochemical differences among strains of a given serotype of Actinobacillus pleuropneumoniae as epidemiological markers, to rapidly identify the source of infection in herds affected with swine pleuropneumonia. Out of 38 different biochemical and physiological tests performed on a total of 67 strains belonging to serotypes 1 and 5 of A. pleuropneumoniae, three fermentation tests, glycerol, lactose and raffinose, allowed the classification of serotype 1 strains into 6 phenotypic groups and serotype 5 strains into 4 of these groups. Groups II and III were exclusively composed of serotype 1 strains, whereas the majority of strains in groups I and IV belonged to serotypes 1 and 5 respectively, the latter comprising almost all the serotype 5 studied.     

Molecular epidemiology of Types I/III strains of Mycobacterium avium subspecies paratuberculosis isolated from goats and cattle

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolates classifies them into three groups: cattle or Type II, sheep or Type I, and intermediate or Type III. To avoid problems associated with characterization of extremely slow growth strains, PCR-based techniques that divide the M. a. paratuberculosis strains in two main groups (cattle or Type II, and sheep or Types I/III) can be performed. The objectives of this study were to characterize the M. a. paratuberculosis isolates identified by different PCR-based tests (IS1311-PCR and restriction endonuclease analysis, PCR test based on a DNA sequence difference, and a PCR aimed at three Type I-specific loci), and to determine the clinical and epidemiological implications of Types I/III M. a. paratuberculosis strains in livestock. One hundred and fifty-eight M. a. paratuberculosis strains from domestic ruminants were analyzed. One hundred and six M. a. paratuberculosis isolates (61 from goats and 45 from cattle) were classified as Type II strains; and 52 (29 from cows, 20 from goats, and three from sheep) were included in the Types I/III. The Types I/III M. a. paratuberculosis strains were associated to Spanish native breeds. The majority of these animals had not been in direct or indirect contact with sheep flocks infected with M. a. paratuberculosis. This fact should be taken into account when implementing paratuberculosis control programs.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

猪、牛源非结核分枝杆菌的分离与鉴定

采集猪颌下淋巴结,猪肠系膜淋巴结,牛颌下淋巴结和牛肠系膜淋巴结各200份,使用改良罗氏培养基进行分离培养和传代培养,通过进行生长特性试验、生化鉴定试验和鉴别培养基生长试验对所分离出的分枝杆菌进行菌型鉴定。结果显示:猪颌下淋巴结中分离出耻垢分枝杆菌4株,鸟分枝杆菌4株,胞内分枝杆菌2株,胃分枝杆菌2株,蟾蜍分枝杆菌1株,龟分枝杆菌龟亚种杆菌1株;猪肠系膜淋巴结未分离出非结核分枝杆菌;牛肠系膜淋巴结分离出瘰疬分枝杆菌2株,加地斯分枝杆菌1株;牛肠系膜淋巴结分离出,瘰疬分枝杆菌5株,金色分枝杆菌2株,戈登分枝杆菌2株,蟾蜍分枝杆菌2株。猪、牛的感染率均为3.5%。     

鸭疫里默氏杆菌分离鉴定及药敏试验

从云南、山东16个鸭场有典型浆膜炎临床症状的病(死)鸭中分离到14株细菌,经形态、培养特性、生化特性、PCR等生物学特性鉴定,上述菌株均为鸭疫里默氏杆菌,型鉴定试验表明其中2株为RA-1型、4株为RA-2型,药敏试验表明山东分离株对头孢氨苄、痢特灵、链霉素、庆大霉素较敏感,云南分离株对氨苄青霉素、痢特灵、头孢氨苄较敏感。动物致病性试验显示RA-1、RA-1型分离菌株均可使健康鸭发病或死亡。     

贵州鸭疫里默氏杆菌的分离鉴定及耐药性分析

从贵州省临床疑似鸭传染性浆膜炎发病鸭鹅中分离病原菌,经细菌分离培养、细菌形态、培养特征及生化试验,初步鉴定出疑似鸭疫里默氏杆菌(Riemerella anatipestifer,RA)20株,经PCR和动物接种试验,其中9株鉴定为RA。对不同地区分离到的这9株菌进行血清型鉴定和15种常规抗菌药物的药敏试验。结果发现,9株RA中4株为血清2型,其余5株暂未鉴定出血清型;9株分离株中,对吡哌酸、链霉素、卡那霉素和红霉素等耐药的菌株比例为80%以上,对先锋霉素高度敏感的菌株比例较高。结果表明,本次分离的RA菌株生化试验鉴定结果与其他地区存在一定差异,不同地区分离到的RA对不同抗菌药的敏感程度存在较大差异性。本研究结果为该地区鸭传染性浆膜炎的药物防制提供了很好的理论依据。     

A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of coagulase-positive staphylococci isolated from bovine milk

A total of 414 coagulase-positive staphylococcal strains obtained at the mastitis laboratory, National Veterinary Institute, Uppsala, Sweden, were studied. One hundred and seventy seven strains were used for a frequency study. Ninety-seven per cent were identified as Staphylococcus aureus, 2% as Staphylococcus intermedius and 1% as Staphylococcus hyicus. Two hundred and thirty seven strains with atypical hemolysis reactions on bovine blood agar were randomly selected, with the aim to increase the number of S. intermedius and S. hyicus strains available for testing. Eight different characteristics, including physiological, enzymatical and biochemical properties, were used to identify the coagulase-positive Staphylococcus species. The results of this study suggest that the following tests should be included for correct identification of the 3 different species of coagulase-positive staphylococci: P agar supplemented with acriflavin, beta-galactosidase and hemolytic reaction on chocolate agar. These 3 tests are simple and quick to perform and enable accurate for easy differentiation of the 3 coagulase-positive Staphylococcus species.     

Molecular epidemiologic features of Corynebacterium pseudotuberculosis isolated from horses

OBJECTIVE: To characterize isolates of Corynebacterium pseudotuberculosis from horses, cattle, and sheep in Colorado, Kentucky, Utah, and California in samples collected during perceived epidemics of infection (increased numbers of cases identified) in 2002 and 2003, and determine how closely isolates were related and their possible source. SAMPLE POPULATION: 54 isolates of C pseudotuberculosis from 49 horses, 4 cattle, and 1 sheep. PROCEDURES: Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) assay, PCR assay for the gene encoding the phospholipase D (PLD) toxin, biochemical analyses, and tests for susceptibility to 17 antimicrobial drugs were performed. RESULTS: All isolates reduced nitrate to nitrite, most yielded positive results for the PLD toxin gene, and all were susceptible to antimicrobial drugs. Ten genetic types were detected by use of RAPD PCR assay; types III to X were isolated from horses, cattle, or both in 1 or more states. Types III and IX were isolated from both horses and cattle. Types VII and VIII were isolated in only 1 state, but the number of isolates in these groups was small. In contrast, all other types were isolated in 2 or more states. All isolates from Utah were type III, but the other 3 states had isolates from more than 1 type. CONCLUSIONS AND CLINICAL RELEVANCE: These data are consistent with a clonally expanding epidemic of infection in Utah and an increase in number of infections caused by multiple strains of C pseudotuberculosis not derived from a single source in the other states. The increase in number of infections could be the result of reporting bias, environmental factors facilitating infection, or host factors such as greater herd susceptibility.     

Biochemical Phenotypes of Salmonella Livingstone Isolated from Humans,Animals and Feedstuffs in Sweden

Salmonella Livingstone is occasionaly isolated from humans, animals and feedstuffs in Sweden. To follow the spread of infection and trace the source of isolates, adequate typing methods are needed. We have developed an automated typing system based on biochemical fingerprinting of bacteria (the PhP system) for typing of different Salmonella serotypes. The system measures the kinetics of various biochemical reactions of bacteria grown in liquid medium in microtiter plates and uses numerical techniques to identify biochemical phenotypes (BPTs) among the tested strains. In the present study we used a set of 16 highly discriminatory tests to differentiate strains of Salmonella of serotype Livingstone and evaluated the system for its discriminatory ability using a collection of 34 unrelated human isolates of S. Livingstone. We also used the system to investigate BPTs of 45 Livingstone strains isolated from animals and feedstuffs in Sweden between 1987 and 1991. Altogether 19 different BPTs were found among human isolate giving a diversity index (Di) of 0.930. In contrast, most strains isolated from animals and feedstuffs in Sweden belonged to 2 dominating BPTs (Di = 0.704). One of these contained 17 strains mainly isolated during 1992 whereas the other contained 18 strains isolated between 1987 and 1991. None of the Swedish human isolates were identical to those of animals and feedstuffs. These findings suggest that 2 different BPTs of Salmonella Livingstone strains are particularly common among animals and feedstuffs in Sweden and that they are not related to human cases of enteritis in this country. We also conclude that biochemical fingerprinting with the PhP system is a reliable and highly discriminatory method for detecting epidemic strains of Salmonella Livingstone.     

基于PFGE技术的酸乳中乳酸菌的基因分型

采用选择性培养基分离酸乳中的保加利亚乳杆菌和嗜热链球菌,并利用生理生化和糖发酵实验结合16S rDNA同源性分析对分离所得菌株进行鉴定,获得保加利亚乳杆菌和嗜热链球菌分离菌株。利用脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分别对保加利亚乳杆菌和嗜热链球菌分离株进行基因分型。结果表明：所检测的酸乳样品中均含有保加利亚乳杆菌和嗜热链球菌;仅有2 种酸乳的嗜热链球菌为同一菌株,其余酸乳中的保加利亚乳杆菌及嗜热链球菌均为不同菌株。PFGE可以对保加利亚乳杆菌和嗜热     

鸭疫里默氏杆菌的分离鉴定

从广州市郊区几个鸭场发病或死亡鸭的脑、心血、肝、脾等组织中分离到6株革兰氏阴性杆菌，经形态学检查、生化试验、动物实验等鉴定均为鸭疫里默氏杆菌。通过凝集试验和琼脂扩散试验鉴定：2株为鸭疫里默氏杆菌Ⅰ型，4株为鸭疫里默氏杆菌Ⅱ型。     

Typing and virulence testing of Pasteurella haemolytica field strains

The greater part of 145 typed Pasteurella haemolytica strains from calf could be attributed to Type A 1. Hence, in the context of virulence testing, this is the most important type at present for calf. Clearly manifest pneumonia was caused in calf by other strains of Types A 2, A 6, A 12, and T 10 which were also tested for their virulence parameters. The same applied to a number of strains which had so far been characterised merely as T or A/T strains.     

致病性鸡大肠杆菌血清型鉴定及多价灭活苗的研制

从新乡市不同鸡场采集大肠杆菌的病料88株,经过分离鉴定,确定出56株为致病性鸡大肠杆菌,血清型分别是O1,O2,O5,O35,O78,O111,其中O1,O2,O78,O111为主要的血清型。将所分离的6种血清型大肠杆菌制成多价灭活苗,接种雏鸡,使实验鸡获得了免疫保护。     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR

Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.     

Characterisation of Rhodococcus equi strains isolated from foals and from immunocompromised human patients

The cultural, morphological, biochemical, serological characteristics and antibiotic susceptibility of 25 Rhodococcus equi strains isolated from lungs and lung abscesses of pneumonic foals and 5 R. equi strains isolated from immuno-compromised human patients were examined. All R. equi strains showed common cultural, morphological and biochemical characteristics both with conventional tests and on the basis of their enzyme profile. The R. equi strains examined were resistant to penicillins with the exception of ampicillin, to sulphamethazine and several strains also to sulphamethoxazole-trimethoprim. All strains were susceptible to erythromycin and rifampicin. The strains isolated from humans showed somewhat higher rate of antibiotic resistance to penicillin, cefotaxime, kanamycin, streptomycin, lincomycin, and oxytetracycline. The overwhelming majority (96%) of the equine isolates belonged to serotype 1 in Prescott's serotyping system, while the human isolates could not be serotyped.     

Cultural and biochemical characteristics of Mycobacterium paratuberculosis isolated from goats in Norway.

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant.The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria.Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

Pathogen Analysis of Goat's Respiratory Diseases

To investigate the pathogens of goat's respiratory disease from a goat farm in Guangxi province, the epidemiological investigation, clinic observation, pathological examination, bacterium isolation and identification, biochemical test, PCR and animal experiment test were conducted.The preventive and control measures were taken on the basis of pathogen epidemiological characteristics and drug sensitive test results.The results showed that Mycoplasma, influenza virus and parasite were negative by isolation and culture or PCR, however, two strains of gram-negative bacteria named MS1 and MS2 were isolated from lung.MS1 was identified as Serratia marcescens by biochemical test and 16S rRNA sequencing, which shared 99% nucleotide homology with other Serratia marcescens in GenBank.And MS2 was identified as E.coil by the same methods that shared 99% nucleotide homology with other E.coli in GenBank.Animal experiment showed that two strains could cause death in mice.The drug sensitive tests showed two strains were highly sensitive to spectinomycin, amikacin, kanamicin and neomycin.Kanamycin and dexamethasone were used to treat the sick goats, and achieved good results.     

山羊呼吸系统疾病病原分析

为了调查广西省某山羊场一起羊呼吸道疾病的病原,本试验采取流行病学调查、临床症状与病理变化观察、病原分离鉴定、生化试验、PCR及致病性试验等方法对病原进行分析,并根据流行病学特点和药敏试验结果进行防控。结果表明,从病死山羊的肺脏组织中分离出MS1和MS2两株革兰氏阴性的致病杆菌,分离培养或PCR检测发现支原体、流感病毒和寄生虫均为阴性。MS1菌株生化特性符合黏质沙雷氏菌,其16S rRNA基因测序结果与GenBank上登录的黏质沙雷氏菌核苷酸序列同源性达到99%以上;MS2菌株生化特性符合大肠杆菌,其16S rRNA基因测序结果与GenBank上登录的大肠杆菌核苷酸序列同源性达到99%以上,两株菌对小白鼠均具有致病性。大肠杆菌和黏质沙雷氏菌均对壮观霉素、阿米卡星、卡那霉素和新霉素高度敏感,用高敏药物卡那霉素联合地塞米松等相关措施进行治疗,收到良好效果。