Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot

Footrot is a debilitating disease of sheep resulting in lameness, production losses and suffering. To study the basic bacteriology of the disease, a survey was initiated across commercial farms and non-commercial research flocks to compare the bacteriology of symptomatic footrot infected sheep with healthy asymptomatic sheep. Of the 80 farmers initially contacted, 14 collected hoof swabs and returned the swabs by post. Following DNA extraction, species-specific PCR was used to identify if Dichelobacter nodosus (D. nodosus) or Fusobacterium necrophorum (F. necrophorum) species were present on each swab. Of the 42 swabs taken from symptomatic footrot infected sheep, 17 were positive for both F. necrophorum and D. nodosus, two were positive for F. necrophorum only, two for D. nodosus only and 23 swabs were negative for both F. necrophorum and D. nod osus. Of the 50 swabs received from healthy asymptomatic sheep, one was positive for F. necrophorum only and 49 were negative for both D. nodosus and F. necrophorum. This suggests that both F. necrophorum and D. nodosus are linked to footrot in the field in a pastoral farming system. If these bacteria are linked together and collectively cause footrot, this may need to be considered when managing a footrot outbreak, or maintaining a quarantine.     

Prevalence of footrot in Swedish slaughter lambs

Background  

Footrot is a world-wide contagious disease in sheep and goats. It is an infection of the epidermis of the interdigital skin, and the germinal layers of the horn tissue of the feet. The first case of footrot in Swedish sheep was diagnosed in 2004. Due to difficulties in distinguishing benign footrot from early cases of virulent footrot and because there is no possibility for virulence testing of strains of Dichelobacter nodosus in Sweden, the diagnosis is based of the presence or absence of clinical signs of footrot in sheep flocks. Ever since the first diagnosed case the Swedish Animal Health Service has worked intensively to stop the spread of infection and control the disease at flock level. However, to continue this work effectively it is important to have knowledge about the distribution of the disease both nationally and regionally. Therefore, the aims of this study were to estimate the prevalence of footrot in Swedish lambs at abattoirs and to assess the geographical distribution of the disease.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

Background  

Chlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.     

The intD mobile genetic element from Dichelobacter nodosus, the causative agent of ovine footrot, is associated with the benign phenotype

The Gram-negative anaerobic pathogen Dichelobacter nodosus is the principal causative agent of footrot in sheep. The intA, intB and intC elements are mobile genetic elements which integrate into two tRNA genes downstream from csrA (formerly glpA) and pnpA in the D. nodosus chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of pnpA. We have proposed a model in which virulence in D. nodosus is controlled in part by the integration of genetic elements downstream from csrA and pnpA, altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the intD element, which is 32 kb in size and contains an integrase gene, intD, several genes related to genes on other integrated elements of D. nodosus, a type IV secretion system and a putative mobilisation region, suggesting that the intD element has a role in the transfer of other genetic elements. Most of the D. nodosus strains examined which contained the intD gene were benign, with intD integrated next to pnpA, supporting our previous observation that virulent strains of D. nodosus have the intA element next to pnpA.     

Development of a real-time multiplex PCR assay for the detection of multiple <Emphasis Type="Italic">Salmonella</Emphasis> serotypes in chicken samples

Background  

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.     

Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients

Background  

Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols.     

A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in <Emphasis Type="Italic">Enterococcus</Emphasis> species

Background  

Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.     

False positive PCR detection of <Emphasis Type="Italic">Tropheryma whipplei</Emphasis> in the saliva of healthy people

Background  

Tropheryma whipplei, the agent of Whipple's disease (WD), has been recently isolated and the genomes of two isolates have been fully sequenced. Previous diagnosis tools for the diagnosis of the disease used sequence analysis of the 16S rRNA gene. Using this target gene, the high percentage of detection of the bacterium in saliva of healthy people was in contrast to the negative results obtained with specific target genes. The aim of our study was to compare previously published primers targeting the 16S rRNA gene to real-time PCR with Taqman* probes targeting specific repeat genes only found in the genome of T. whipplei in a series of 57 saliva from healthy people.     

A longitudinal study of <Emphasis Type="Italic">Campylobacter</Emphasis> distribution in a turkey production chain

Background  

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis. Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.     

High throughput detection of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> by real-time PCR with internal control system and automated DNA preparation

Background  

Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated.     

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.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.     

Quick PCR based diagnosis of typhoid using specific genetic markers

A quick multiplex PCR based detection method was developed for early diagnosis of typhoid using specific genetic markers of S. typhi. Primers of tyv gene, flag gene, viaB gene and ratA gene confirmed the specificity and sensitivity of the PCR. The serum samples of the suspected typhoid patients were taken directly for PCR without culturing and isolating genomic DNA. Overall diagnosis required 2 h which is the least time ever reported for a PCR based methods. The sensitivity of the method is up to 5 fg genomic DNA. The genetic markers are specific and the four pairs of primers give selective amplification and differentiate S. typhi from closely related S. typhimurium.     

PCR and real-time PCR primers developed for detection and identification of <Emphasis Type="Italic">Bifidobacterium thermophilum</Emphasis>in faeces

Background  

Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.     

Rapid <Emphasis Type="Italic">Leptospira</Emphasis> identification by direct sequencing of the diagnostic PCR products in New Caledonia

Background  

Most of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment.     

Rapid differentiation of <Emphasis Type="Italic">Francisella</Emphasis> species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

Background  

Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.     

Development of real-time PCR for detection of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

Real‐time PCR for Helicobacter pylori diagnosis. The best tools available

Background

Knowledge of antimicrobial susceptibility, especially to macrolides, has become crucial for the management of Helicobacter pylori infection. Our aim was to evaluate two new PCR kits able to detect H. pylori in gastric biopsies as well as the mutations associated with macrolide resistance.

Materials and Methods

Two hundred successive biopsies (received from gastroenterologists all over France) were used. The two new kits tested were Amplidiag H. pylori+Clari^R from Mobidiag Espoo, Finland, and RIDA^®GENE H. pylori from R‐Biopharm, Darmstadt, Germany. Culture and a validated in‐house real‐time PCR were also performed, and in the case of a positive culture, Etest for clarithromycin was carried out. Discrepancies were solved by looking at the pathologic data.

Results

Culture was positive in 68 cases (34%), and with our in‐house real‐time PCR in these 68 cases plus 5 others (N = 73, 36%). All were also detected by the two new kits. In addition, RIDA^®GENE H. pylori detected one more positive also detected by Amplidiag H. pylori+Clari^R, and Amplidiag detected two other positives. Of these three additional cases, pathology confirmed the positivity for two. Only one case diagnosed by Amplidiag could be considered as a false positive. With regard to clarithromycin resistance, 22 cases were detected. The corresponding mutations (A2142/43G) were all identified with the three PCRs.

Conclusions

These two new kits which have an excellent sensitivity and specificity are convenient to use, adaptable to different thermocyclers, provide quick results, and deserve to be used in H. pylori diagnosis for a better choice of treatment regimen.