Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly β-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.     

相同耐药谱的11株大肠杆菌的脉冲场电泳分型研究

为了了解同一猪场不同来源具相同耐药谱的11株大肠杆菌之间的同源性,以探讨其耐药流行机制,用脉冲场凝胶电泳(PFGE)对同一猪场不同来源具相同耐药谱的大肠杆菌做染色体DNA分型.结果发现分别来自小猪和环境的2株大肠杆菌通过PFGE分型,属于同一个亚型.初步认为不同来源的大肠杆菌来自不同克隆系,同时在动物环境之间可能存在着耐药性的克隆传播.结果表明,PFGE可作为耐药性流行病学调查的一种研究方法.     

Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis

Background

Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.

Results

The isolates (n = 185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.

Conclusions

In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.     

Construction and characterization of a BAC library for sunflower (Helianthus annuus L.)

A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.     

Distribution of Campylobacter jejuni isolates from turkey farms and different stages at slaughter using pulsed-field gel electrophoresis and flaA-short variable region sequencing

The aim of this study was to assess the diversity of thermotolerant Campylobacter spp. isolated from turkey flocks at six rearing farms 1-2 weeks prior to slaughter (360 faecal swab samples) and from 11 different stages at the slaughterhouse (636 caecal, environmental, neck skin and meat samples). A total of 121 Campylobacter isolates were identified to species level using a multiplex PCR assay and were typed by pulsed-field gel electrophoresis (PFGE) and flaA-short variable region (SVR) sequencing. All Campylobacter isolates were identified as Campylobacter jejuni. PFGE analysis with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA SVR typing yielded in nine flaA-SVR alleles. The Campylobacter-positive turkey flocks A, C and E were colonized by a limited number of Campylobacter clones at the farm and slaughter. The present study confirms the traceability of flock-specific strains (PFGE types I, V and IX; flaA types 21, 36 and 161) from the farm along the entire processing line to meat cuts. It seems that stress factors such as high temperature of the defeathering water (54-56 °C), drying of the carcass skin during air chilling (24 h at 2 °C), and oxygen in the air could not eliminate Campylobacter completely. Campylobacter-negative flocks became contaminated during processing by the same subtypes of Campylobacter introduced into the slaughter house by preceeding positive flocks even if they were slaughtered on subsequent days. Proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination, especially in countries with a low prevalence of Campylobacter spp. The majority of flaA SVR alleles displayed a distinct association with a specific PFGE type. However, a linear relationship for all strains among both typing methods could not be established. To specify genetic relatedness of strains, a combination of different genotyping methods, is needed.     

Surveillance of verocytotoxigenic Escherichia coli in Irish bovine dairy herds

Verocytotoxigenic Escherichia coli (VTEC) are highly significant zoonotic threats to public health, and have been the causative agent implicated in numerous high-profile outbreaks affecting large numbers of people. Serovar O157 is most frequently linked with human illness; however, other serovars, such as O26, O103, O111 and O145, have also been implicated. This study aimed to characterize the prevalence and virulence determinants of these five serovars in Irish dairy farm herds, and their milk. Using real-time PCR (RTi-PCR), bovine rectal faecal swabs and raw milk samples, along with milk filters, were screened for the presence of vt genes. Positive samples were then screened for the five serovars using sero-specific PCR. Serovar-positive samples were subjected to immunomagnetic separation, to isolate viable VTEC strains. These isolates were subsequently screened for four virulence factors: vt1, vt2, eaeA and hlyA. Three hundred and eighty six of the 600 rectal faecal swabs, 85 of the 117 milk-filters and 43 of the 120 bulk-tank milk samples, were positive for vt genes. From these 514 total vt-positive samples, 58 O26, 162 O103, 1 O111, 324 O145 and 26 O157 positives were detected by sero-specific RTi-PCR. Immunomagnetic separation yielded 12 O26, 26 O103, 0 O111, 19 O145 and 10 O157 isolates. Ten of these isolates contained at least one of the four virulence determinants screened for (i.e. vt1, vt2, eaeA and hlyA). Of these 10 isolates, pulsed-field gel electrophoresis showed that two of the O26 isolates from different farms were indistinguishable. Two O157 isolates were also indistinguishable. This study found serovars O103 and O145 to be the most prevalent in samples tested. Apart from the occurrence of VTEC in dairy herds, this study shows a high occurrence of vt genes in the environment, creating the possibility of horizontal gene transfer and emergence of new VTEC strains.     

Occurrence of ɛ-proteobacterial species in rabbits (Oryctolagus cuniculus) reared in intensive and rural farms

In order to investigate the occurrence of Campylobacter, Helicobacter and Arcobacter species in caecal contents of rabbits reared in intensive and rural farms, a total of 87 samples from animals belonging to 29 farms were analysed by both cultural and PCR analyses.     

Genetic analysis of clinical and vaccine strains of Bordetella pertussis by Pulsed-Field Gel Electrophoresis (PFGE), Multi Locus Sequence Typing (MLST) and serotyping

In spite of high vaccination coverage in the Expanded Program of Immunization (EPI), pertussis has not been eradicated yet and the re-emergence of the disease is still reported worldwide. The genetic divergence study of circulating clinical strains of Bordetella pertussis among the population with high vaccination coverage is a useful tool to have an insight in the understanding of genetic patterns of this bacterium and deviation of them from vaccine strains. Different methods are accessible for studying of Bordetella pertussis that can perform appropriate assessment between populations. Strains used in this study were a collection of two pertussis vaccine strains used to create killed pertussis vaccine over years at Razi Vaccine and Serum Research Institute, 10 clinical and 2 reference strains (ATCC9797 and Tohama I) in Multilocus Sequence Typing (MLST), Pulsed-Field Gel Electrophoresis (PFGE), and serotyping. The genetic profiles of vaccine working and master seeds showed no important change(s) in frequencies of fingerprint types investigated in the vaccine strains and had homogeneity in PFGE method where the clinical isolates showed diversity in genetic profile. Serotyping method showed that all of 10 clinical strains expressing Fim 3. In MLST study, seven housekeeping genes including adk, pgm, fum C, tyr B, gly A, pep A and icd were analyzed which showed no changes in the sequence of clinical and vaccine strains with 100% homology. The genes that cause pathogenicity like ptxC, tcfA and fhaB were also evaluated and the results illustrated heterogeneity in the vaccine and circulating strains.     

Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms

The main objective of the present study was to investigate if different kinds of pig farms, like farrowing farms and rearing farms, play a role in the transmission of methicillin-resistant Staphylococcus aureus (MRSA) to Dutch finishing farms. Twelve farrowing farms, 11 finishing farms, 6 farrow-to finish farms, 1 rearing farm and 1 centre for artificial insemination were included. Screening of 310 pigs from these 31 farms showed 35 pigs (11%) to carry MRSA in their nares. On 7 of the 31 (23%) investigated farms colonized pigs were found, including 3 finishing farms, 3 farrowing farms and 1 farrow-to-finish farm. The use of standard antimicrobial medication of the pigs seemed to be a risk factor for MRSA carriage. Screening of the pigs on six farms supplying pigs for the MRSA positive farms revealed that the pigs on all but one farm were MRSA positive. Genotyping revealed that all MRSA strains were non-typeable by PFGE using the SmaI restriction enzyme and had multilocus sequence type (MLST) ST398. Different spa-types were found including t011, t108, t567, t899 and t1939, but the spa-types on epidemiologically related farms were identical indicating that MRSA are transmitted between farms through the purchase of colonized pigs. Two SCCmec types were found among the MRSA: type IV and type V. SCCmec type V was predominant. On two farms MRSA isolates with ST398, the same spa-type but with different SCCmec types (IV and V) were found, suggesting that different SCCmec elements have been inserted into MSSA with the same genotype. All MRSA strains were resistant to tetracycline, but additional resistances to erythromycin, lincomycin, kanamycin and gentamicin were also found. All MRSA isolates were negative for the exfoliative toxin genes (eta and etb), PVL toxin genes (lukF and lukS), toxic shock syndrome gene (tst-1), and the leukotoxin genes (lukE, lukD, lukM, lukF').     

四川省一些实验动物养殖场SPF大鼠和小鼠肠内容物中金黄色葡萄球菌的检测

调查四川省一些实验动物养殖场的实验动物中金黄色葡萄球菌(Staphylococcus aureus,SA)携带情况,找出传播途径和进化规律,分析其污染原因,为保障实验动物质量提供技术支撑。按照GB/T 14926.14-2001《实验动物金黄色葡萄球菌检测方法》对2011年-2017年四川省部分实验动物中SA进行检测,对分离的SA菌株进行PCR耐药基因mecA检测,并应用金黄色葡萄球菌A蛋白(Staphylococcus protein A,SPA)和脉冲场电泳(pulsed field gel electrophoresis,PFGE)进行分子分型。结果显示,2011年-2017年共从7家实验动物养殖场采集并完成370只实验动物中SA的检测,共检出SA 31株,总分离率为12.16%,且均从SPF大鼠中分离所得。31株SA的mecA基因均为阴性。SPA可分成5个型别,其中T2360为优势型别,PFGE型别有10个带型。四川省养殖实验动物中仅SPF大鼠携带SA,不同年份检出SA基因型别基本不同,不同养殖场同一年检出的SA具有相同基因型,说明不同养殖场中的SA可能来自同一污染源,应加强SPF大鼠监控,以确保实验动物的质量。