Validation of a real‐time PCR for Haemophilus parasuis

Aims: To validate a real‐time PCR test for the diagnosis of Glässer’s disease, a major pig disease caused by Haemophilus parasuis. Methods and Results: The specificity of a real‐time PCR amplifying the inf B gene was validated with 68 H. parasuis isolates and 36 strains of closely related species. As well, 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were tested with the real‐time PCR, and the results were compared with culture and a conventional PCR. The real‐time PCR produced significantly more positive results than the conventional PCR (165 vs 86). Conclusions: The sensitivity of the real‐time PCR combined with high specificity makes it a very valuable tool for the diagnosis of Glässer’s disease. Significance and Impact of Study: This new method will improve the ability of laboratories to diagnose Glässer’s disease, especially in laboratories where the culture method for H. parasuis is not optimal.     

Genome sequence of Haemophilus parasuis strain 29755

Haemophilus parasuis is a member of the family Pasteurellaceae and is the etiologic agent of Glässer’s disease in pigs, a systemic syndrome associated with only a subset of isolates. The genetic basis for virulence and systemic spread of particular H. parasuis isolates is currently unknown. Strain 29755 is an invasive isolate that has long been used in the study of Glässer’s disease. Accordingly, the genome sequence of strain 29755 is of considerable importance to investigators endeavoring to understand the molecular pathogenesis of H. parasuis. Here we describe the features of the 2,224,137 bp draft genome sequence of strain 29755 generated from 454-FLX pyrosequencing. These data comprise the first publicly available genome sequence for this bacterium.     

副猪嗜血杆菌血清4、13和14型TbpA对豚鼠的交互免疫保护性

【背景】副猪嗜血杆菌(Haemophilusparasuis,HPS)是猪革拉瑟氏病(Gl?sser?sdisease)的病原体,目前在对该病的防控中,由于长期使用抗生素产生的耐药性以及灭活疫苗缺乏交互免疫保护力,亟待寻求新的方法来解决这一问题。【目的】探究不同血清型HPS转铁结合蛋白A (TbpA)对豚鼠的交互免疫保护性,为进一步用仔猪开展相关研究奠定基础。【方法】采用HPS血清型4、13和14型重组TbpA以0.1mg/只、经2次间隔期为20d的免疫后,检测豚鼠血清IgG抗体和细胞因子(IL-2、IL-5、IL-8、IFN-γ、MCP-1、TNF-α)水平;以HPS血清型4、13和14型菌株5 LD50剂量腹腔攻毒,检查病理组织学变化及其免疫保护率。【结果】HPS血清型4、13和14型重组TbpA均能诱导豚鼠产生较高水平的特异性抗体IgG,并使细胞因子显著升高。HPS血清型4、13和14型重组TbpA在免疫后均能对豚鼠产生交互免疫保护,其中HPS血清型13型TbpA免疫组的交互免疫保护率最高,对血清型4、14型HPS均为50.0%,对相同血清型HPS的免疫保护率也最高,达到83.3%,病理组织学检查显示与HPS血清型4、14型TbpA免疫组相比,13型TbpA免疫组的组织切片病理变化与攻毒对照组之间差异更明显。【结论】HPS血清型4、13和14型TbpA均能诱导豚鼠的体液免疫和相关细胞因子的分泌,产生交互免疫保护力,其中HPS血清型13型Tbp A的交互免疫保护力最强,可作为一种新的疫苗候选抗原。     

安徽PRSS发病猪副猪嗜血杆菌的分离鉴定及其耐药性分析

目的了解猪繁殖与呼吸综合征（PRRS）发病猪继发感染副猪嗜血杆菌（HPS）的情况及分离菌株的药物感受性。方法应用细菌分离培养、形态学检查、生化试验和PCR技术,对2007年1月至2008年12月从安徽不同地区采集的146头份PRRS发病猪的病料进行HPS检测,并采用标准K-B纸片法对分离菌株进行14种抗菌药物敏感试验。结果分离鉴定出12株HPS,检出率为8.22%（12/146）;12株HPS对氯霉素100%敏感,环丙沙星为91.7%,阿莫西林和新霉素为83.3%,对罗红霉素100%耐药,阿米卡星为83.3%。结论安徽省不同地区PRRS感染猪群中均存在程度不同的HPS感染,各地区HPS分离株表现出形态上的变化特征和一致的生化特性,且有对临床常用抗菌药物耐药性增强的趋势。     

Development of a real‐time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs

Aims: To investigate whether the relative abundance of the Bacteroidetes and Firmicutes divisions in pigs is different between obese and lean animals. Methods and Results: Group‐specific primers were designed to target the 16S rRNA genes of Bacteroidetes and Firmicutes present in the gut. After the validation of their specificity, these primers were used in the real‐time PCR quantification of all Bacteria, Firmicutes division, Bacteroidetes division and Bacteroides spp. in the faecal samples of obese and lean pigs from Banna mini‐pig inbred line. The obese pigs had a ～61% fewer percentage (based on all Bacteria) of Bacteroidetes division (P = 0·033) and a ～56% fewer proportion of Bacteroides spp. (P = 0·047) than the lean pigs. The proportions of both Bacteroidetes and Bacteroides had a negative correlation (P < 0·01) with the body weight. Conclusion: The results suggested that the fat storage might affect the proportion of Bacteroidetes division in the gut. Significance and Impact of the Study: The real‐time PCR assays developed for Firmicutes and Bacteroidetes will be useful for investigating the composition of gut microbiota.     

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis

Haemophilus parasuis is the causative agent of Gl?sser's disease, which is responsible for considerable economic losses in the pig-rearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpB-V5-His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availability.     

Comparison of loop-mediated isothermal amplification and real-time PCR for the diagnosis of tuberculous pleurisy

Aims: Tuberculous pleurisy is an important cause of pleural effusions in areas with a high incidence of tuberculosis. In this study, we developed an IS1081‐based LAMP for the detection of Mycobacterium tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy. Methods and Results: Investigation of pleural effusion samples from patients with tuberculous pleurisy, majority of them smear‐/culture‐negative, and control individuals with non‐TB diseases showed that the LAMP assay with incubation time of 60 min has much higher specificity and the LAMP assay with incubation time of 90 min has significantly higher sensitivity in the diagnosis of tuberculous pleurisy, as compared with fluorescent real‐time PCR. Conclusions: The MTBC–LAMP is a useful assay for the diagnosis of tuberculous pleurisy, especially in pleural effusion smear‐/culture‐negative patients. Significance and Impact of the Study: Tuberculous pleural effusion usually contains low number of mycobacteria, which leads to low diagnostic sensitivity of acid‐fast staining and mycobacterial culture methods. In this study, we developed a simple and sensitive LAMP assay for the diagnosis of tuberculous pleurisy. This assay should have broad application in resource‐limited settings.     

Quantitative analysis of cutaneous malassezia in atopic dermatitis patients using real-time PCR

We quantified the cutaneous Malassezia in patients with atopic dermatitis using a real-time PCR assay. Seven to 12 times more Malassezia colonized the head and neck compared to the trunk or limbs, and the species M. globosa and M. restricta accounted for approximately 80% of all Malassezia colonization at any body site.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR

Aims: In this article, a quantitative real‐time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time‐consuming and not always accurate. Methods and Results: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non‐target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C_t values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10–14 CFU ml^?1 in either cola or beer and at levels of 9·4–25·0 CFU ml^?1 in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. Conclusions: The results indicate that real‐time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. Significance and Impact of the Study: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.     

Characterization of the inhabitancy of mouse intestinal bacteria (MIB) in rodents and humans by real-time PCR with group-specific primers

Mouse intestinal bacteria (MIB) is a new operational taxonomic unit (OTU) belonging to the Bacteroides subgroup in the Cytophaga-Flavobacter-Bacteroides (CFB) phylum recently found in the intestine of mice, rats and humans. However, their characters are still unknown since they have not yet been isolated by culture. To understand their habitat characteristics in intestinal tracts, the quantification assays of MIB were established using MIB group-specific primers. The MIB population in the intestine was evaluated as a percentage of the number of 16S rRNA gene copy of MIB. A real-time PCR assay using group specific primers showed the fluctuation of MIB inhabitancy and revealed that the MIB population in the small intestine of mice was significantly lower than the large intestinal contents. Moreover, MIB was found in human feces though the number was lower than in murine. This assay using group-specific primers revealed new information about host-preference of MIB.     

Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR

A second gene encoding a functional tryptophan hydroxylase activity has recently been described (TPH2), which is expressed abundantly in brainstem, the primary site of serotonergic neurons in the CNS. As serotonin (5-HT) has an important role as a precursor of the nocturnal synthesis of the pineal gland hormone, melatonin, it was of interest to determine the relative expression of TPH1 and 2 mRNA in the rat pineal during the light:dark (L:D) cycle using sensitive real-time RT-PCR assays which were developed for each TPH isoform. TPH1 mRNA expression was 105-fold more abundant in rat pineal than TPH2, and showed a significant approximately 4-fold nocturnal increase in expression which may contribute to the previously described nocturnal increase in pineal tryptophan hydroxylase activity. TPH2 expression within the gland showed no significant variation with time of day and was very low (approximately 300 copies/gland) indicating expression in the small proportion of "non-pinealocyte" cells in the gland.     

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis

A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.     

Quantification of algal endosymbionts (Symbiodinium) in coral tissue using real-time PCR

Understanding the flexibility of the endosymbioses between scleractinian corals and single‐cell algae of the genus Symbiodinium will provide valuable insights into the future of coral reefs. Here, a real‐time polymerase chain reaction (PCR) assay is presented to accurately determine the cell densities of Symbiodinium clades C and D in the scleractinian coral Acropora millepora, which can be extended to other coral–symbiont associations in the future. The assay targets single‐ to low‐copy genes of the actin family of both the coral host and algal symbiont. Symbiont densities are expressed as the ratio of Symbiodinium cells to each host cell (S/H ratio, error within 30%), but can also be normalized to coral surface area. Greater accuracy in estimating ratios of associations involving multiple clades is achieved compared with previous real‐time PCR assays based on high‐copy ribosomal DNA loci (error within an order of magnitude). Healthy adult A. millepora containing ~1.4 × 10⁶ zooxanthellae per cm² (as determined by haemocytometer counts) had S/H ratios of c. 0.15, i.e. ~15 symbiont cells per 100 host cells. In severely bleached colonies, this ratio decreased to less than 0.005. Because of its capacity to accurately determine both densities and ratios of multiple symbionts within one sample, the assay will open the door for novel research into the mechanisms of symbiont shuffling and switching.     

Population dynamics and in situ kinetics of nitrifying bacteria in autotrophic nitrifying biofilms as determined by real-time quantitative PCR

Population dynamics of ammonia-oxidizing bacteria (AOB) and uncultured Nitrospira-like nitrite-oxidizing bacteria (NOB) dominated in autotrophic nitrifying biofilms were determined by using real-time quantitative polymerase chain reaction (RTQ-PCR) and fluorescence in situ hybridization (FISH). Although two quantitative techniques gave the comparable results, the RTQ-PCR assay was easier and faster than the FISH technique for quantification of both nitrifying bacteria in dense microcolony-forming nitrifying biofilms. Using this RTQ-PCR assay, we could successfully determine the maximum specific growth rate (mu = 0.021/h) of uncultured Nitrospira-like NOB in the suspended enrichment culture. The population dynamics of nitrifying bacteria in the biofilm revealed that once they formed the biofilm, the both nitrifying bacteria grew slower than in planktonic cultures. We also calculated the spatial distributions of average specific growth rates of both nitrifying bacteria in the biofilm based on the concentration profiles of NH4+, NO2-, and O2, which were determined by microelectrodes, and the double-Monod model. This simple model estimation could explain the stratified spatial distribution of AOB and Nitrospira-like NOB in the biofilm. The combination of culture-independent molecular techniques and microelectrode measurements is a very powerful approach to analyze the in situ kinetics and ecophysiology of nitrifying bacteria including uncultured Nitrospira-like NOB in complex biofilm communities.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

Proportion of prey consumed can be determined from faecal DNA using real-time PCR

Reconstructing the diets of pinnipeds by visually identifying prey remains recovered in faecal samples is challenging because of differences in digestion and passage rates of hard parts. Analysing the soft-matrix of faecal material using DNA-based techniques is an alternative means to identify prey species consumed, but published techniques are largely nonquantitative, which limits their usefulness for some applications. We further developed and validated a real-time PCR technique using species-specific mitochondrial DNA primers to quantify the proportion of prey in the diets of Steller sea lions (Eumetopias jubatus), a pinniped species thought to be facing significant diet related challenges in the North Pacific. We first demonstrated that the proportions of prey tissue DNA in mixtures of DNA isolated from four prey species could be estimated within a margin of ～ 12% of the percent in the mix. These prey species included herring Clupea palasii, eulachon Thaleichthyes pacificus, squid Loligo opalescens and rosethorn rockfish Sebastes helvomaculatus. We then applied real-time PCR to DNA extracted from faecal samples obtained from Steller sea lions in captivity that were fed 11 different combinations of herring, eulachon, squid and Pacific ocean perch rockfish (Sebastes alutus), ranging from 7% to 75% contributions per meal (by wet weight). The difference between the average percentage estimated by real-time PCR and the percentage of prey consumed was generally <12% for all diets fed. Our findings indicate that real-time PCR of faecal DNA can detect the approximate relative quantity of prey consumed for complex diets and prey species, including cephalopods and fish.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.