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During routine electron microscopy of fecal samples from diarrheic dogs dated from 2000 virus particles resembling circovirus in shape and size were detected in two samples (V2177/00; V3374/00). Polymerase chain reaction (PCR) using primers specific for porcine circovirus type 2 (PCV2) amplified DNA recovered from both samples. Sequencing of PCR amplificates (V2177/00) obtained with PCV2-specific primer pairs revealed a genome size of 1768 bp. The nucleotide sequence was highly similar (98% nucleotide identity) to the PCV2a reference sequence., Highlights • Electron microscopy has revealed circoviruses in two fecal samples of diarrheic dogs. • PCV2-specific PCR amplified DNA extracted from the samples. • By sequencing one sample a genome size of 1768 bp and a nucleotide sequence identity of 98% to PCV2a were achieved. • This is the first time that PCV2 has been reported in dogs.

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s–2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates.

International audience; We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

Porcine circovirus 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome (PMWS), has been detected in North American and European wild boars at prevalences arguing for high circulation rates among populations. Systematic data on the qualitative distribution of PCV2 infections and on PCVD (PCV2 diseases) in wild boars are rare, however, and quantitative data about viral loads are missing. To be able to judge the PCV2/PCVD situation in wild boars, evaluation of the nationwide qualitative and quantitative distribution of PCV2 and PCVD in Germany was the objective of the present study. Wild boar samples were compared with domestic pig samples of the same greater areas, including tonsils, lungs, spleen, Lnn. bronchiales and Lnn. mesenterici of 349 wild boars and 348 domestic pigs. All of the wild boars and 308 of the domestic pigs have been apparently free of PCVD, 40 of the domestic pigs had been rejected from slaughter due to health problems (i.e. wasting). Tissues were examined by pathohistology, immunohistology (IHC), nested PCR (nPCR and quantitative PCR (qPCR). One wild boar (0.3%) and 8.7% of the domestic pigs were classified as PCVD-affected, based on pathohistology and IHC. PCV2 DNA was detected in 63.1% and 45.4% of the wild boars by nPCR and qPCR, respectively, and in 100% and 98.8% of the domestic pigs. PCV2 loads differed significantly between wild boars (average: 10(2.8) PCV2 genomes/microg extracted sample DNA) and domestic pigs (average: 10(4.2) PCV2 genomes/microg of sample DNA). The qualitative detection of PCV2 DNA in tissues of wild boars and domestic pigs was abundant and not of any pathological relevance. The overall load of PCV2 in domestic pigs was relatively high and borderline with respect to PCVD, and there was no difference between apparently healthy pigs and pigs rejected from slaughter in this respect. Most of the wild boars were infected with PCV2 at loads less relevant for PCVD.

Beside domestic pigs wild boars can also be affected by postweaning multisystemic wasting syndrome (PMWS). For the first time a nationwide survey of wild boars (n = 356) and domestic pigs (n = 340) was carried out in Germany by histopathology, immunohistochemistry (IHC) and quantitative PCR (qPCR). Whereas 102/340 domestic pigs were immunoreactive for PCV2 antigen in at least one examined tissue, only 8/356 wild boars reacted positively. Similar findings could be found in qPCR: all domestic pigs showed viral DNA in at least one tissue, while in the examined tissues of 170 wild boars PCV2-DNA was not detectable. The specimens were examined histologically for histiocytosis and depletion of lymphocytes, both typical for PMWS. Based on these findings, six wild boars and 69 domestic pigs were assumed to be affected by PMWS.

PCV-2 is involved in “postweaning multisystemic wasting syndrome” (PMWS), “porcine dermatitis and nephropathy syndrome” (PDNS), respiratory and reproductive disorders, and thereby plays a crucial role in today's swine production world-wide. The virus is apparently ubiquitous in domestic pigs and has also been demonstrated in wild pigs. Up to now, a characterization of PCV-2 samples from wild pigs, which might help to estimate the possible role of wild pigs as sources of domestic pig infection, has not been carried out. Spleen samples from 16 PCV-2-positive wild pigs from hunting grounds of four regions in Germany were used for the analysis of the viral genome. In one sample, the complete sequence of the genome was determined. In the other, a 742 nucleotide fragment from the highly variable capsid sequence of the ORF2 was sequenced. Analysis of the sequences led to the identification of three PCV-2 strains. One strain, representing 14 of the 16 samples, was closely related with Chinese, but not with German strains. The genome of this strain was shortened by one nucleotide by a deletion close to the end of ORF2. The deletion led to a shift of the stop-codon and to the insertion of a further codon. Two further strains differed in up to 4.7% of nucleotides and up to 10.5% of amino acids (aa). These strains were aligned with clusters of PCV-2 samples from mainly French and German domestic origin.

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.

Mycobactin J-dependent mycobacterial isolates from sheep, goat, and cattle herds with Johne's disease in Morocco, South Africa, the United States, and Germany were tested for the repetitive insertion sequence IS900 of Mycobacterium paratuberculosis by PCR. The IS900 PCR target sequence was detected in 90 of 93 fecal culture isolates tested (96.8%). Restriction fragment length polymorphisms (RFLPs) and in vitro growth characteristics were studied in 46 of the IS900-positive isolates and in two bovine vaccine strains of M. paratuberculosis. Five different RFLP types were identified in PvuII digests of genomic DNA by Southern hybridization with a DNA probe specific for IS900. All isolates of M. paratuberculosis could be classified into two major clusters by their growth rates as well as the relatedness of their PvuII-RFLP hybridization patterns. All of the sheep isolates were classified into cluster I (extremely slow growth), while all cattle and goat isolates were members of cluster II (moderately slow growth). Different PvuII-RFLP patterns were detected in different sheep flocks from Morocco and South Africa. Our results demonstrate that genetically and phenotypically different strains of M. paratuberculosis were present in ruminant populations. The strains from sheep in Morocco and South Africa tested in the study appeared to belong to a unique group of M. paratuberculosis strains that might have adapted to this host species. The presence of several genetically distinct strains in different sheep flocks suggested that analysis of IS900-specific RFLP patterns may provide a useful tool for the epidemiologic investigation of ovine paratuberculosis outbreaks.

Actinobacillus pleuropneumoniae is an important pathogen of the porcine respiratory tract. Subclinically and chronically infected pigs may enter a permanent carrier state, harboring A. pleuropneumoniae in lungs or tonsils. Despite the enormous role of A. pleuropneumoniae in domestic swine production, no published data about prevalence and distribution of A. pleuropneumoniae infections in wild boars (Sus scrofa) are available. Antibodies against A. pleuropneumoniae have been detected in serum of wild boars from Slovenia. The aim of the present study was to evaluate A. pleuropneumoniae infection status in wild boars in Germany. Tissue samples from lungs and tonsils of 531 wild boars from 52 hunts during the hunting seasons 2004-05 to 2006-07 were examined for A. pleuropneumoniae by polymerase chain reaction (PCR). Although gross pathologic lesions typical for pleuropneumonia were not detected, 35.8% of the wild boars were PCR-positive. Tonsils were involved in 94.3% and lungs in 15.5%. Actinobacillus pleuropneumoniae infection prevalence increased with age and body weight. Further research is needed to understand the involvement of different A. pleuropneumoniae serotypes and to understand the interrelationship of A. pleuropneumoniae infection between wild boars and domestic pigs.

A multiplex real-time PCR assay was developed to detect and quantify B. hyodysenteriae, B. pilosicoli, and L. intracellularis in pig faeces. Specific probes and primers were directed against the NADH oxidase (nox) gene of Brachyspira and the aspartate ammonia lyase (aspA) gene of L. intracellularis, respectively. The analytical sensitivity for the real-time PCR assay, expressed as limit of detection (LOD) was below 10 DNA copies for L. intracellularis, 14 DNA copies for B. pilosicoli and 26 DNA copies per PCR reaction for B. hyodysenteriae. The experimental sensitivity, expressed as limit of quantitation (LOQ) for the real-time PCR assay was set to 100 DNA copies per PCR reaction which equals 8 x 10(3) cells per gram of faeces. The multiplex real-time PCR was tested in parallel to conventional PCR on 749 faecal samples from 121 farms. 73 (9.7%), 30 (4%), and 30 (4%) faecal samples were positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively by conventional PCR and 59 (7.9%), 27 (3.6%), and 7 (0.9%) by multiplex real-time PCR. From the real-time PCR positive results 34 (4.5%), 25 (3.3%), and 4 (0.5%) were above the LOQ. The multiplex real-time PCR will allow rapid and quantitative detection of clinical relevant amounts of the three key porcine enteric pathogens simultaneously.

Comparative genomic analysis of a wild-type strain of the ovine pathogen Chlamydophila abortus and its nitrosoguanidine-induced, temperature-sensitive and virulence-attenuated live vaccine derivative identified point mutations unique to the mutant (Burall et al. [1]). Here, we evaluate the capacity of some of these mutations to either create or eliminate restriction sites using the wild-type strain C. abortus S26/3 as a reference. Three of eight genomic sites with confirmed point mutations (CAB153, CAB636 and CAB648) were retained for analysis as each resulted in the loss of a restriction site in the genome sequence of the vaccine strain. PCR-restriction fragment length polymorphism analysis using restriction enzymes chosen to specifically target the three genomic sites was then applied to a large number of C. abortus field isolates and reference strains. Our results indicate that the three mutations are uniquely present in the vaccine strain, and as such provide easy-to-use markers for the differential identification of the vaccine strain and wild-type isolates.

The article contains sections titled: 1. Abbreviations and Glossary of Special Terms 2. Introduction 3. Principle 4. Reaction Components 4.1. DNA Polymerases 4.1.1. Fidelity 4.1.2. Mg2+ as Cofactor 4.1.3. Inhibitors 4.2. Deoxynucleoside Triphosphates (dNTPs) 4.3. Reaction Buffer 4.4. Primer 4.4.1. Melting Temperature 4.4.2. Concentration 4.4.3. Synthesis 4.4.4. Primer Design 4.5. Template 5. Equipment and Laboratory Preconditions 6. Cycling Conditions and Reaction Phases 6.1. Denaturation Conditions 6.2. Annealing Conditions 6.3. Extension Conditions 6.4. Amplification Phases 7. Characterization of PCR Products 7.1. Gel Electrophoresis 7.2. Restriction Analysis 7.3. Hybridization 7.4. Sequencing 8. Modifications of PCR for Increased Specificity and Sensitivity 8.1. Increasing Specificity 8.2. Increasing Sensitivity 9. Contamination 9.1. Sources of Contamination 9.2. PCR Controls 9.3. Decontamination Methods 9.3.1. Uracil-N-Glycosylase (UNG) 9.3.2. UV Light 9.3.3. Enzymes 9.3.4. Psoralens and Isopsoralens 10. Quantitative PCR 10.1. External Control 10.2. Internal Control 10.3. Real-Time Quantification 10.3.1. Fluorescence Resonance Energy Transfer (FRET) 10.3.2. Forster Energy Transfer (TaqMan System) 11. Applications 11.1. Detection of Pathogenic Agents 11.1.1. Preconditions 11.1.2. Extraction of Template DNA 11.1.2.1. Isolation of Total DNA 11.1.2.2. Isolation of Pathogen Specific DNA 11.1.3. Amplification 11.1.4. Multiplex PCR 11.1.5. In Situ PCR 11.1.6. Universal PCR 11.1.7. PCR Controls for Diagnostic Purposes 11.2. Detection of Genetic Disorders or Cancerous Cells 11.2.1. Known Mutations 11.2.1.1. Amplification Refractory Mutation System (ARMS) 11.2.1.2. Allele-Specific Oligonucleotides (ASOs) 11.2.1.3. Introduction of Restriction Sites 11.2.1.4. Competitive PCR 11.2.1.5. Primer Extension PCR (PEST) 11.2.2. Unknown Mutations 11.2.2.1. Denaturing Gradient Gel Electrophoresis (DGGE) 11.2.2.2. Single Strand Conformation Polymorphism (SSCP) 11.2.2.3. Chemical Cleavage of Mismatches (CCM) 11.3. PCR Analysis in Evolution and Taxonomy 11.3.1. Restriction Fragment Length Polymorphism 11.3.2. PCR for Typing Satellites or HLA Genes 11.3.3. Random Amplified Polymorphic DNA 11.3.4. Analysis of Fingerprints 11.3.5. Restrictions and Future Aspects 11.4. Tissue Typing 11.5. PCR in Food Analysis 11.6. PCR to Engineer DNA 11.6.1. Cloning of PCR Products 11.6.2. PCR Mutagenesis 11.7. PCR for Analysis of Gene Expression

Although Brachyspira (B.) hyodysenteriae and Lawsonia (L.) intracellularis are widely distributed in pigs in Germany, there exists limited information on their clinical relevance. To get more insight into their potential role in swine diarrhoeal disease, in 2002 and 2003 faecal specimens from healthy pigs (n=1445) as well as from diarrhoeic pigs (n=2002) were tested by polymerase chain reaction (PCR) for the presence of both agents. Of the specimens from healthy pigs L. intracellularis and B. hyodysenteriae were detected in 7.3% and 6.7%, respectively. In contrast, of the diarrhoeic pigs the ratios of positive samples amounted to 19.4% for L. intracellularis and 17.9% for B. hyodysenteriae. Concerning the age of the diseased animals, in growing pigs the detection rates of L. intracellularis and B. hyodysenteriae were nearly identical (16.4% and 14.2%, respectively). In fattening pigs a significant higher number of animals were affected with B. hyodysenteriae (35.8%) than with L. intracellularis (28.2%). On the other hand, in sows L. intracellularis (35.6% positive samples) was dominant compared to B. hyodysenteriae (21.2% positive samples). Considering the nearly threefold higher percentage rates of L. intracellularis and B. hyodysenteriae in diarrhoeic pigs in comparison to healthy pigs, it is concluded that both agents play an important role in swine diarrhoeal disease. The results further indicated that in fattening pigs B. hyodysenteriae and in sows L. intracellularis have a dominant role, respectively.

Strains and isolates of Coxiella burnetii, an obligate intracellular bacterium, carry a single plasmid or a plasmid-homologous sequence integrated into the chromosome. The plasmids QpH1, QpRS, QpDV and the chromosome-integrated plasmid-homologous region have been completely sequenced, whereas no sequence data are available for the QpDG plasmid. In this study, we used total genomic DNA from reference strain C. burnetii Dugway 5J108-111 to demonstrate and characterize the QpDG plasmid by pulsed-field gel electrophoresis (PFGE) and Southern hybridization. Primers derived from regions shared among C. burnetii plasmids were used to construct a physical map of the QpDG plasmid by extra long (XL) PCR. Both approaches, Southern hybridization and XL PCR indicated that QpDG and QpH1 represent a closely related and likely identical plasmid.

Candida isolates (n=23) derived from Testudinidae were investigated by multiplex-polymerase chain reaction (PCR). The isolates comprised 13 Candida (C.) tropicalis, nine C. albicans and one C. parapsilosis. In addition, five reference strains C. albicans (ATCC 10231), C. tropicalis (DSM 4238), C. parapsilosis (DSM 4237), C. glabrata (ATCC 70614) and C. krusei (ATCC 70075) were investigated. PCR revealed easily distinguishable species-specific amplicons of the chs1-gene and resulted in a clear identification in all cases. No discrepancies between conventional identification techniques and the PCR-based system were seen. The described PCR offers a rapid alternative to conventional techniques for the identification of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.