Colonisation of pet shop puppies with Brachyspira pilosicoli

Anaerobic intestinal spirochaetes of the genus Brachyspira are known to colonise dogs, but relatively little is known about their prevalence, distribution or pathogenic potential. One species, Brachyspira pilosicoli, is thought to cause diarrhoea in dogs, as well as in other animals and humans. To investigate the prevalence and distribution of infection, faecal samples from 49 puppies from six pet shops in the suburbs of Perth, Western Australia were subjected to selective culture for anaerobic intestinal spirochaetes. Growth from the primary plates was also harvested, the DNA extracted and a polymerase chain reaction (PCR) amplification of a portion of the 16S rRNA gene of B. pilosicoli applied. Weakly beta-haemolytic intestinal spirochaetes (WBHIS) grew on plates from 20 of the dogs (40.8%). Seven plates (14.2%) yielded PCR positive amplification for B. pilosicoli. Seven WBHIS isolates were obtained in pure culture, and two of these were shown to be B. pilosicoli by PCR. Application of multilocus enzyme electrophoresis to the seven isolates confirmed that the two PCR positive isolates were B. pilosicoli, whilst the other five belonged to a group previously designated "Brachyspira canis". All the "B. canis" isolates came from healthy puppies, suggesting that this WBHIS is a commensal. Three of the seven puppies with PCR evidence of B. pilosicoli had diarrhoea, but the sample size was small and the association between colonisation and diarrhoea was not statistically significant. Pet shop puppies are commonly infected with intestinal spirochaetes, and may act as a reservoir of B. pilosicoli for other animals and humans.     

Detection of Lawsonia intracellularis,Serpulina hyodysenteriae,weakly beta-haemolytic intestinal spirochaetes,Salmonella enterica,and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs

A polymerase chain reaction (PCR) was optimized to detect Lawsonia intracellularis in faeces from naturally infected pigs. By combining a boiling procedure to extract DNA and a nested PCR procedure, a detection limit at 2×10² bacterial cells per gram of faeces was achieved. The optimized PCR was used together with conventional culture techniques to detect Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes (WBHIS), Salmonella enterica, and haemolytic Escherichia coli, in a case control study to examine selected risk factors for the development of diarrhoea in growing pigs. Herds with diarrhoea were selected as cases and randomly chosen herds without diarrhoea were chosen as controls. Infection with L. intracellularis significantly enhanced the chance of diarrhoea. S. hyodysenteriae, WBHIS group IV (Serpulina pilosicoli), and S. enterica were isolated only from case herds which indicate that these species may influence the development of diarrhoea. In addition, herd-type had a significant impact, that is specific pathogen-free herds showed an odds ratio at 0.2 relative to conventional herds for the development of diarrhoea.     

Identification and differentiation of canine Mycoplasma isolates by 16S-23S rDNA PCR-RFLP

Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the "classic" non-hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species.     

Molecular identification of Erysipelothrix isolates from the tonsils of healthy cattle by PCR

For 79 isolates from the tonsils of healthy cattle identified as Erysipelothrix by cultivation, biochemical and serological tests, genotypic identification was performed by polymerase chain reaction (PCR) using four species-specific sets of oligonucleotide primers (ER1F-ER1R, ER2F-ER2R, ER3F-ER3R and ER4F-ER4R). The results of PCR for 79 bovine isolates were compared with those of serological typing. For 19 isolates, serotyping and genotyping results were the same. PCR allowed for the identification of 36 untypable isolates as Erysipelothrix species, strain 1. Serotyping and genotyping results of the remaining 24 isolates were different. Supplemental tests are frequently needed for Erysipelothrix identification.     

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.     

Detection of urease-negative Bordetella bronchiseptica from the field

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs? assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.     

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.     

New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples

Chlamydophila psittaci and Chlamydophila abortus are the causative agents of avian chlamydiosis (psittacosis) and ovine enzootic abortion, respectively. Both pathogens are known to possess zoonotic potential. Due to their close genetic relatedness, direct and rapid species identification is difficult. In the present study, new real-time PCR assays are reported for both species. The tests are based on highly specific probes targeting the ompA gene region and were conducted as duplex PCRs including an internal amplification control. The Cp. psittaci assay successfully passed a proficiency test at national level. Examination of field samples revealed Cp. psittaci as the dominating species in birds, but also Cp. abortus in a few psittacines. Real-time PCR assays for species-specific detection of Cp. psittaci and Cp. abortus are suited for routine diagnosis, which renders them important tools for the recognition of outbreaks of psittacosis and ovine enzootic abortion.     

PCR-based differentiation of three porcine Eimeria species and Isospora suis

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.     

仲彬草属物种细胞质基因组PCR-RFLP分析

应用PCR-RFLP技术对32份仲彬草属和2份外类群共34份材料细胞质基因组DNA进行遗传变异分析。选用的16个引物中4个叶绿体引物和5个线粒体引物可扩增出稳定而明显的PCR产物,其扩增产物用15种限制性内切酶进行酶切。结果发现,所有的135种引物/酶组合中,得到清晰稳定的83种引物/酶组合,其中在32份仲彬草属材料中有40种引物/酶组合(占48.2%)能检测到多态性,但多态性水平较低。32份仲彬草属物种间的遗传相似系数在0.833以上,平均值为0.903。聚类分析表明,细胞质基因组PCR-RFLP标记能将全部供试材料区分开,32份仲彬草属材料聚为4类。同种不同居群细胞质间的遗传变异很小,分别聚在一起;种间的细胞质遗传差异大于种内不同居群间的遗传差异;而形态相似、地理分布一致的物种有聚类在一起的倾向。因此,利用细胞质基因组PCR-RFLP标记不仅可检测到仲彬草属种间多态性,也可检测到种内多态性。细胞质基因组PCR-RFLP标记能为仲彬草属物种系统学研究提供有价值的资料。     

Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profiles, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identification of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confirm phenotypic identification of colonies using species-specific primers, capsule type using serogroup-specific primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1-53% of tissue samples collected from pigs 5-6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confirmed as P. multocida using a species-specific PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specific, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.     

Molecular assay for the identification of Setaria tundra

We report the molecular characterization of the amplification products obtained by specific PCR experiments aimed to identify the 5S ribosomal spacer of Setaria tundra specimens isolated from roe deer (Capreolus capreolus) in north Italy, which represent the first record of the parasite in this country. Three different fragments of approximately 400, 800, and 1200 bp (base pairs) are produced. Sequence analyses showed that all three fragments share a very high level of similarity to 5S spacer sequences of some Setaria species and other filariae present in genebank. Based on these sequences, we were able to design species-specific PCR primers for the precise identification of S. tundra.     

Improved polymerase chain reaction technique for determining the species composition of Eimeria in poultry litter

An improved polymerase chain reaction (PCR)-based method for determining the species composition of Eimeria in poultry litter was developed by incorporating species-specific internal standards in the assay. Internal standard molecules were prepared by fusing seven different Eimeria species-specific intervening transcribed sequence 1 (ITS1) rDNA primer pairs to a non-Eimeria DNA molecule and by cloning the hybrid DNA molecules into a plasmid. The internal DNA standards were then used in Eimeria-specific ITS 1 PCR, and they were found to be capable of detecting E. acervulina, E. maxima, E. praecox, and E. tenella oocysts isolated directly from poultry litter.     

Identification by culture, PCR, and immunohistochemistry of mycoplasmas and their molecular typing in sheep and lamb lungs with pneumonia in Eastern Turkey

This study used cultures, polymerase chain reaction (PCR), and immunoperoxidase to examine samples from 216 lungs from sheep and lambs with macroscopic pneumonia lesions for the presence of Mycoplasma species. DNA was extracted from lung tissue samples and broth cultures with the help of a DNA extraction kit and replicated using genus-specific and species-specific primers for mycoplasma. The lung samples were examined by the immunoperoxidase method using hyperimmune Mycoplasma ovipneumoniae serum. The randomly amplified polymorphic DNA (RAPD) test was used for the molecular typing of M. ovipneumoniae isolates. Mycoplasma was isolated in the cultures of 80 (37.03 %) of a total of 216 lung samples. Genus-specific mycoplasma DNA was identified by PCR in 96 (44.44 %) samples in broth cultures and 36 (16.66 %) directly in the lung tissue. Of these 96 cases in which genus-specific identification was made, 57 (59.37 %) were positive for reaction with species-specific primers for M. ovipneumoniae and 31 (32.29 %) for Mycoplasma arginini. The DNA of neither of the latter two species could be identified in the remaining eight samples (8.33 %) where mycoplasma had been identified. As for the immunoperoxidase method, it identified M. ovipneumoniae in 61 of 216 lung samples (28 %). Positive staining was concentrated in the bronchial epithelium cell cytoplasm and cell surface. RAPD analysis resulted in 15 different profiles. Our results suggest that PCR methods could be successfully used in the diagnosis of mycoplasma infections as an alternative to culture method and identifying this agent at the species level.     

Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe

Biting midges of the Culicoides obsoletus Meigen and Culicoides pulicaris L. species complexes (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palearctic regions. However, predicting epidemiological risk and the spread of disease is hampered because whilst vector competence of Culicoides is expressed only in adult females, morphological identification of constituent species is only readily applicable to adult males and some species distinguishing traits have overlapping character states. Furthermore, adult males are typically rare in field collections, making characterisation of Culicoides communities impossible. Here we highlight the utility of mitochondrial cytochrome oxidase subunit I (COI) DNA sequences for taxonomic resolution and species identification of all species within C. obsoletus and C. pulicarus complexes. Culicoides were collected from 18 sites in the UK and Continental Europe, and identified to species level, or species complex level, based on morphological characters. The sample comprised four species from the C. obsoletus complex (n = 88) and five species from the C. pulicaris complex (n = 39). The DNA sequence of the 5' end of the COI gene was obtained from all individuals. Each member species formed a well-supported reciprocally monophyletic clade in a maximum likelihood phylogeny. Levels of DNA sequence divergence were sufficiently high between species to allow the design of species-specific PCR primers that can be used in PCR for identification of members of the C. pulicaris complex or in a multiplex PCR to identify members of the C. obsoletus complex. This approach provides a valuable diagnostic tool for monitoring species composition in mixed field collections of Culicoides.     

Detection of Mycoplasma bovis with an improved pcr assay

A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml(-1) in broth cultures using ethidium-bromide-stained agarose gels.     

Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.     

用PCR和PCR-RFLP方法检测和鉴定进口海鱼中的异尖线虫幼虫

应用PCR和PCR-RFLP方法对厦门口岸进口的各种海鱼中的异尖线虫幼虫进行检测鉴定。结果在带鱼、竹荚鱼、金线鱼、大眼鲷和白姑鱼等海鱼中发现了简单异尖线虫、典型异尖线虫、对盲囊线虫和针回线虫等4种异尖线虫。带鱼和竹荚鱼的异尖线虫感染率达100%,竹荚鱼异尖线虫感染强度最高,白姑鱼感染率和感染强度相对较低。少数海鱼同时感染2种以上异尖线虫,从带鱼中同时检出简单异尖线虫和针蛔线虫,从竹荚鱼中检出简单异尖线虫、对盲囊线虫和典型异尖线虫。内切酶实验结果表明,应用限制性内切酶HinfI有效鉴别上述4种异尖线虫。     

Occurrence and species level diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in healthy and diarrheic dogs and cats

In order to study the occurrence and co-infection of different species of Campylobacter, enteric Helicobacter and Anaerobiospirillum in dogs and cats and define a possible association between these microrganisms and gastrointestinal disorders, 190 dogs and 84 cats, either healthy or with diarrhea, were sampled between 2002 and 2003. Thirty-three C. upsaliensis, 17 C. jejuni, 2 C. helveticus, 1 C. lari isolates from dogs and 14 C. helveticus, 7 C. jejuni, 6 C. upsaliensis isolates from cats were identified using species-specific PCR and phenotypic tests. Whole cell protein profile analysis, phenotypic tests, PCR-RFLP of gyrB and a phylogenetic study of partial groEL and 16S rRNA sequences were used to identify 37 H. bilis, 22 H. canis and 14 H. cinaedi in dogs and 12 H. canis, 5 H. bilis and 2 H. cinaedi in cats. Whole cell protein profile analysis, phenotypic tests and species-specific PCR of 16S rRNA were used to identify 14 A. succiniciproducens, 12 A. thomasii isolates and one unidentified Anaerobiospirillum sp. isolate in dogs and 3 A. thomasii isolates in cats. Fifty-two animals (19%) were positive for the isolation of more than one genus. No significant statistical correlation was found between any isolates of Campylobacter, Helicobacter or Anaerobiospirillum spp. or the various co-infection rates, and the presence of diarrhea in either dogs or cats. Campylobacter isolates were also tested for antibiotic resistance using the agar dilution method.     

Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene

On the basis of a species-specific PCR assay, a RFLP analysis for typing of Haemophilus parasuis strains was developed and evaluated. Amplification was based on the gene tbpA, encoding a transferrin-binding protein. RFLP analysis of the 1.9-kb tbpA-amplicon using TaqI, AvaI and RsaI endonucleases produced 12 different patterns for the reference strains of the 15 known H. parasuis serovars, and showed a high heterogeneity (33 RFLP groups) for 101 H. parasuis clinical isolates tested. The sensitivity, typeability (100% versus 65% for immunodiffusion), high degree of discrimination (0.93 versus 0.84 for immunodiffusion), simplicity and low cost per test make this PCR-RFLP assay a useful method for typing H. parasuis and, therefore, for studying the epidemiology of outbreaks of Gl?sser's disease.