Molecular detection of Streptococcus agalactiae in bovine raw milk samples obtained directly from bulk tanks

Mastitis is the most common infectious disease affecting dairy cattle; in addition, it remains the most economically important disease of dairy industries around the world. Streptococcus agalactiae, a contagious pathogen associated with subclinical mastitis, is highly infectious. This bacterium can cause an increase in bulk tank bacterial counts (BTBC) and bulk tank somatic cell counts (BTSCC). The microbiological identification of S. agalactiae in samples from bulk tanks is an auxiliary method to control contagious mastitis. Thus, there are some limitations for time-consuming cultures or identification methods and additional concerns about the conservation and transport of samples. Bulk tank samples from 247 dairy farms were cultured and compared through polymerase chain reaction (PCR), directed to 16S rRNA genes of S. agalactiae, followed by BTBC and S. agalactiae isolation. The mean value of BTBC was 1.08 x 10(6) CFU mL(-1) and the bacterium was identified through the microbiological method in 98 (39.7%; CI95% = 33.8-45.9%) and through PCR in 110 (44.5%; CI95% = 38.5-50.8%) samples. Results indicated sensitivity of 0.8571 +/- 0.0353 (CI95% = 0.7719-0.9196) and specificity of 0.8255 +/- 0.0311 (CI95% = 0.7549-0.8827). The lack of significant difference between microbiological and molecular results (kappa = 0.6686 +/- 0.0477 and CI95% = 0.5752-0.7620) indicated substantial agreement between the methods. This suggests that PCR can be used for bulk tank samples to detect contagious mastitis caused by S. agalactiae. (C) 2011 Elsevier Ltd. All rights reserved.

Actinobaculum suis Detection Using Polymerase Chain Reaction

Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR) for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0 x 10(1) CFU/mL and 1.0 x 10(2) CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192) in sow urine samples and 82.2% (37/45) in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45) of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs.

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

Climate and infectious disease: Use of remote sensing for detection of Vibrio cholerae by indirect measurement

It has long been known that cholera outbreaks can be initiated when Vibrio cholerae, the bacterium that causes cholera, is present in drinking water in sufficient numbers to constitute an infective dose, if ingested by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish water may directly or indirectly depend on such conditions as water temperature, nutrient concentration, and plankton production that may be favorable for growth and reproduction of the bacterium. Although these environmental parameters have routinely been measured by using water samples collected aboard research ships, the available data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both expensive and time-consuming. Interpolation to regional scales can also be problematic. Although the bacterium, V. cholerae, cannot be sensed directly, remotely sensed data can be used to infer its presence. In the study reported here, satellite data were used to monitor the timing and spread of cholera. Public domain remote sensing data for the Bay of Bengal were compared directly with cholera case data collected in Bangladesh from 1992–1995. The remote sensing data included sea surface temperature and sea surface height. It was discovered that sea surface temperature shows an annual cycle similar to the cholera case data. Sea surface height may be an indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was also found to be correlated with cholera outbreaks. The extensive studies accomplished during the past 25 years, confirming the hypothesis that V. cholerae is autochthonous to the aquatic environment and is a commensal of zooplankton, i.e., copepods, when combined with the findings of the satellite data analyses, provide strong evidence that cholera epidemics are climate-linked.

Development of PCR-based markers for detection of Leifsonia xyli subsp. xyli in fibrovascular fluid of infected sugarcane plants

DNA of Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease of sugarcane, was detected in the fibrovascular fluid of sugarcane plants using random amplified polymorphic DNA PCR-based amplification using two 10-mer oligonucleotide primers. The primers OPC-02 and OPC-11 produced Lxx-specific markers of approximately 800 bp and 1000 bp, respectively. A cloned DNA fragment from the 800 bp PCR product (pSKC2-800) hybridised to a single genomic DNA fragment from Lxx when used as a probe in Southern hybridisation. This cloned fragment did not hybridise to L. xyli subsp. cynodontis (Lxc), or L. xyli-like bacteria isolated from grasses in Australia, indicating the usefulness of this DNA fragment as a specific probe for Lxx. A cloned fragment from the 1000 bp PCR product ( pSKC11-1000) hybridised to three genomic fragments in Lxx isolates, one genomic fragment in two of the four isolates of L. xyli-like bacteria, and in two of the four isolates of Lxc isolated from the USA. These results indicate that L. xyli-like bacteria are more likely to be related to Lxc than Lxx. These probes did not hybridise to the DNA from strains of the species of Clavibacter, Rathayibacter, Acidovorax, Ralstonia, Pseudomonas and Xanthomonas tested. Two oligonucleotide primers (21-mer) designed from the pSKC2-800 sequences specifically amplified template DNA from Lxx and detected as few as 5 x 10(4) cells/mL in fibrovascular fluid from sugarcane plants infected with Lxx.

The detection of environmental autoinducing peptide quorum-sensing genes from an uncultured Clostridium sp in landfill leachate reactor biomass

Aims: To elucidate whether a dominant uncultured clostridial (Clostridium thermocellum-like) species in an environmental sample (landfill leachate), possesses an autoinducing peptide (AIP) quorum-sensing (QS) gene, although it may not be functional. Methods and Results: A modified AIP accessory gene regulator (agr)C PCR protocol was performed on extracted DNA from a landfill leachate sample (also characterized by 16S rRNA gene cloning) and the PCR products were cloned, sequenced and phylogenetically analysed. It appeared that two agrC gene phylotypes existed, most closely related to the C. thermocellum agrC gene, differing by only 1 bp. Conclusions: It is possible to specifically identify and characterize the agrC AIP QS gene from uncultured Firmicutes (C. thermocellum-like) bacteria derived from environmental (landfill leachate) sample. Significance and Impact of the Study: This is the first successful attempt at identifying AIP QS genes from a cellulolytic environment (landfill). The agrC gene was identified as being most closely related to the C. thermocellum agrC gene, the same bacterium identified as being dominant, according to 16S rRNA gene cloning and subsequently fluorescence in situ hybridization analyses, in the same biomass.

Development of a whole cell biochip for toxicant detection.

In the development of biosensors for ecotoxicity testing it is desirable to produce a small, portable system that can be used in the field. Toxicity testing using bioluminescence is widely used in the laboratory utilising natural and genetically modified (lux/ luc-marked) bacteria and other microorganisms. It is currently not possible to use genetically manipulated microorganisms in field testing and a biosensor, therefore, that incorporates naturally luminescent organisms may be preferred. In the development of a biosensor it is aimed to use the naturally luminescent bacterium Vibrio fischeri as a toxicity detection system on a chip. The bacterium will be immobilised in a polymeric matrix. Current work deals with the optimisation of light output and light preservation within the bacterium prior to immobilisation in polyvinyl alcohol. An examination of a range of physicochemical conditions within the polymer will be made, including cell density, thickness of polymer film, growth and light induction environment, and, preservation conditions, in order to develop a testing system giving consistent results over the lifetime of the biosensor. Data will be presented on light production using different culture media for the growth of V. fischeri and retention of light under immobilised conditions. .