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1.
Principal aim of this study was to examine fecal samples from pigs suffering from diarrhea for the presence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli. The molecular techniques such as PCR and nested PCR were employed to detect the presence of p78 fragment of genomic DNA specific for Lawsonia intracellularis as well as fragment of tlyA gene specific for Brachyspira hyodysenteriae and 16S rDNA gene of Brachyspira pilosicoli. We assumed that about 25% of pigs were infected with Lawsonia intracellularis, about 10% with Brachyspira hyodysenteriae and only 0,8% with Brachyspira pilosicoli. In about 3% mixed infection with L. intracellularis and B. hyodysenteriae was observed. Results were comparable in herds that differed in quantity, breeding technology, hygienic standards and preventive treatment with different chemotherapeutics.  相似文献   

2.
Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.  相似文献   

3.
Lawsonia (L.) intracellularis, Brachyspira (B.) hyodysenteriae and B. pilosicoli are important pathogens in domestic pig production world-wide, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. Conventional PCR is the major diagnostic tool in the detection of the three pathogens, but the sole detection of bacterial DNA might lead to misinterpretations of results with respect to their clinical relevance, especially with mixed infections. Thus, the present study targeted the detection and quantification of the three pathogens in samples from herds with a case history of diarrhoea. Herds and samples were selected by the practitioners on a voluntary basis. Results were based on 1176 individual samples from 95 herds from Southern Germany. The pathogens were detected simultaneously by multiplex real-time PCR. The overall prevalence for L. intracellularis, B. hyodysenteriae and B. pilosicoli was 12.6%, 8.4% and 3.2% in faecal samples and 48.4%, 24.2% and 31.6% in herds, respectively. Sixty one percent, 82.6%, and 73.4% of herds positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively, had mixed infections. Median log values of DNA equivalents/g of faeces for L. intracellularis, B. hyodysenteriae and B. pilosicoli were 3.3, 5.9 and 3.2, with maxima of 8.3, 8.0 and 6.3, respectively. Within herd prevalence of B. hyodysenteriae and B. pilosicoli as well as the load of B. hyodysenteriae were significantly associated with the severity of diarrhoea.  相似文献   

4.
The aim of the present study was to survey the prevalences of the enteric pathogens Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in Swedish growing pigs and in the Swedish wild boar population and to relate these findings to clinical signs. The study included 105 randomly selected herds, constituting approximately one third of Swedish herds with a herd size of >100 sows. The herds were located all over the country. In these herds, growth promoters were not used and pigs sampled were not subjected to any medication. From each herd, samples were taken from 10 growing pigs aged 8-12 weeks, corresponding to approximately 2.5% of all growing pigs present in the herd at the sampling occasion. If possible, the samples were taken from pigs with diarrhoea. Forty-eight faecal samples and 71 rectal swabs were also taken from free-living wild boars (31 piglets, 19 growers and 21 adult animals) at shooting. The samples were analysed by culture and biochemical tests for the presence of Brachyspira spp. and by nested PCR for the presence of L. intracellularis. Brachyspira hyodysenteriae was not demonstrated in any sample. Brachyspira intermedia was detected in 22 samples originating from 15 herds, Brachyspira innocens/Brachyspira murdochii was detected in 370 samples from 82 herds and B. pilosicoli was detected in 134 samples originating from 34 herds. In 21 herds and in 534 samples, no Brachyspira spp. were detected. Lawsonia intracellularis was demonstrated in 285 samples from 50 herds. Further, 418 samples from conventional herds were negative with respect to L. intracellularis and in 345 samples the PCR had been inhibited. All samples from the wild boars were negative for Brachyspira spp., 12 of 48 samples were negative for L. intracellularis, and in 36 wild boar samples, the PCR was inhibited.  相似文献   

5.
Diarrhoea in growing and finishing pigs is usually caused by infectious agents and laboratory diagnosis is a prerequisite for efficient therapy. Cultivation of Brachyspira hyodysenteriae or Brachyspira pilosicoli and detection of Lawsonia intracellularis by means of immunofluorescence tests (IFT) are time-consuming and in some cases lack sensitivity. A multiplex-PCR was designed to detect simultaneously these three pathogens in faeces and tissue samples, allowing the differential diagnosis of dysentery, intestinal spirochaetosis and proliferative enteropathy. Detection limits for B. hyodysenteriae, B. pilosicoli and L. intracellularis were 10(4), 10(2) and 10(3) copies respectively. Agreement between multiplex-PCR and nested-PCR or cultivation was considered substantial to almost perfect. Agreement between multiplex-PCR and IFT in detecting L. intracellularis was only moderate, which was probably related to false-positive results given by IFT. The multiplex-PCR described herein is a valuable tool for the rapid and simultaneous detection of three different pathogens in porcine samples causing enteric diseases.  相似文献   

6.
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.  相似文献   

7.
Faeces samples were taken three times at two-week intervals, from the farrowing units of four herds of known Brachyspira (formerly Serpulina) status and one of unknown Brachyspira status. Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira group III were isolated from the faecal samples from the weaners in the herds using either a maximum of 50 ppm of olaquindox or no feed additives. The detection rates were relatively consistent. However, B hyodysenteriae was not detected at one sampling in a known positive herd. The prevalence of Brachyspira species was also studied in feeder pigs originating from LSO 2000 health class farrowing units, comparable with specific pathogen-free herds. These farms were free from swine dysentery, sarcoptic mange, swine enzootic pneumonia and progressive atrophic rhinitis. Fifty of 428 herds were sampled once. B hyodysenteriae was not isolated from any of them, but B intermedia, B pilosicoli and Brachyspira group III were isolated from five, 14 and 37 of the herds, respectively. The detection of Brachyspira species did not relate to the prevalence of diarrhoea in the herds, as judged by the farmers. The herds using carbadox (40 to 50 ppm) had a lower prevalence of Brachyspira species than those using olaquindox (40 to 50 ppm).  相似文献   

8.
A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.  相似文献   

9.
Pathogenic intestinal spirochaetes of pigs include Brachyspira (formerly Serpulina) hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic spirochetosis (PCS). The purpose of this study was to assess the relative importance of Brachyspira species in diarrhoeal disease of growing pigs on farms in southern Brazil. The intensity and pattern of haemolysis, the production of indole and the hydrolysis of hippurate by reference and field porcine intestinal spirochaetes were compared with 16S-ribosomal RNA (mRNA)- and 23S-rRNA-based polymerase chain reaction assays for the identification of B hyodysenteriae and B pilosicoli. Between July and October 1998, 206 rectal swabs were taken from pigs on 17 farms with a history of diarrhoea developing within 30 days after they had been moved from nursery to growing facilities. Of 49 beta-haemolytic spirochaetes that were cultured, 29 (59.2 per cent) were grown in pure culture for phenotypic and genotypic characterisation, leaving 20 untyped. Of the 29 typed isolates, eight isolates obtained from six farms were identified as B hyodysenteriae, and 15 isolates obtained from seven other farms were identified as B pilosicoli; the remaining six isolates were identified as weakly beta-haemolytic commensal spirochaetes. There was complete agreement between the results of the phenotypic and genotypic analyses.  相似文献   

10.
The aim of this study was to obtain prevalence estimates about the most important enteropathogenic bacteria: Lawsonia intracellularis, Brachyspira hyodysenteriae, Brachyspira pilosicoli, Salmonella enterica and Clostridium perfringens A and C in Hungarian farrow-to-finish pig herds. A total of 31 herds were selected, from where six pooled faecal samples, each containing three individual rectal faecal samples were collected from fattening pigs of 5-6 months of age. All 186 samples were examined by polymerase chain reaction (PCR) for the presence of the pathogens mentioned above. Lawsonia intracellularis was found in 29 herds (93.55%) and in 108 samples (58.06%); B. hyodysenteriae in 14 herds (45.16%) and in 23 samples (12.37%); B. pilosicoli in 19 herds (61.29%) and in 53 samples (28.49%); S. enterica in 17 herds (54.83%) and in 40 samples (21.50%). We detected the presence of C. perfringens A in 19 herds (61.29%) and in 46 samples (24.73%), while C. perfringens C was found in 8 herds (25.81%) and in 11 samples (5.91%). All examined herds were infected with one or more of these agents. Herds with diarrhoea in the mid- to late finishing phase had almost 10 times higher prevalence of B. hyodysenteriae than herds without such a history.  相似文献   

11.
We observed a significant difference in the organic acid profile of diarrheal feces between pigs infected with and free from pathogenic spirochetes. Diarrhea and loose feces were collected from growing pigs, held at 15 different commercial farms. A total of 106 samples were measured for organic acid concentration by HPLC and were checked for the presence of B. hyodysenteriae and B. pilosicoli by PCR. B. hyodysenteriae was detected in 3 samples collected from one farm. B. pilosicoli was detected in 5 samples collected from another farm. Lower concentrations of iso-butyrate and iso-valerate were likely associated with development of pathogenic spirochete infection.  相似文献   

12.
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.  相似文献   

13.
The survival of Brachyspira hyodysenteriae and Brachyspira pilosicoli was investigated at 10 degrees C in laboratory microcosms consisting of soil, porcine faeces, and in soil mixed with 10% porcine faeces, respectively. By plate spreading, survival of B. hyodysenteriae was found to be 10, 78 and 112 days in soil, soil mixed with 10% faeces, and in porcine faeces, respectively. The identities of the colonies on the plates were confirmed using PCR targeting 23S rDNA for specific detection of B. hyodysenteriae. A positive PCR signal could be obtained up to 112 days in all microcosms by direct extraction of DNA from microcosms followed by PCR.The survival time for B. pilosicoli was 119 days in pure soil and 210 days in soil mixed with 10% porcine faeces and in pure faeces, respectively, as determined by plate spreading followed by PCR. On the other hand, by direct extraction of DNA followed by specific detection by PCR. B. pilosicoli could be detected up to 330 days in all microcosms.Dot blot hybridisation with digoxigenin-labelled specific oligonucleotide probe targeting rDNA could not be used for direct detection of Brachyspira spp. from microcosms due to low sensitivity. However, it was used for confirmation of the identity of colonies and proved to be a useful technique.These results show that the two Brachyspira species may survive in outdoor environment for the times shown in these investigations using laboratory microcosms.  相似文献   

14.
The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.  相似文献   

15.
A survey is given on the occurrence and distribution of different Brachyspira species in pigs, in the northwest of Germany. In total 2975 specimen (feces, fecal swabs, colon) were taken and sent for laboratory analysis during the years 1997 to 1999. 1218 Brachyspira (B.) strains were found by cultural analysis. 1757 samples (59%) were negative. The cultural and biochemical differentiation revealed 720 (59.1%) strains B. hyodysenteriae (77.5% were indole negative), 22 (1.8%) B. pilosicoli, 29 (2.4%) B. intermedia, 167 (3.7%) B. innocens and 114 (9.4%) B. murdochii. 166 (13.6%) strains could not be identified. These strains could either not be compared with any of the described species by the methods used or it was impossible to achieve a pure culture from these isolates. The results demonstrate the wide spread of B. hyodysenteriae in pig herds in the northwest of Germany with a very high prevalence of indole negative strains. The most frequent strain was B. hyodysenteriae. B. pilosicoli which causes spirochaetal diarrhoea was rarely isolated and seems not to play an important role in Germany. Experience from routine cultures for Brachyspira give evidence that it is more useful to examine faeces from single pigs instead of pooled samples from a herd. It is recommended to use special transport media for the transport of the specimen.  相似文献   

16.
Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.  相似文献   

17.
The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological-biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.  相似文献   

18.
The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.  相似文献   

19.
The epidemiology of infection with the intestinal spirochaete Brachyspira pilosicoli within pig herds is incompletely understood. To investigate this further, cross-sectional and cohort studies were undertaken on two piggeries. Faeces were subjected to selective culture, and DNA was extracted from growth on the primary media and amplified by polymerase chain reaction (PCR). On one farm, samples from other animal species and the environment were also examined. Isolates were subjected to multilocus enzyme electrophoresis (MLEE) and pulsed field gel electrophoresis (PFGE). The prevalence on farm A (>2000 sows) was 2.4% (95% confidence interval (CI): 0.3, 4.4%). Infection was largely confined to grower/finisher pigs. The six isolates of B. pilosicoli recovered belonged to a single MLEE electrophoretic type (ET) and a single PFGE type. On piggery B, an 80-sow unit located on a research farm, the prevalence amongst growers and finishers was 12.2% (95% CI: 4.7, 19.6%). There was also evidence that weaners were being infected. Ten isolates obtained were genetically heterogeneous, being divided into six ETs and seven PFGE types. One of four isolates in one ET had an identical PFGE type to those on piggery A, and may have been introduced to piggery B in stock from piggery A. On farm B, B. pilosicoli was also detected by PCR in chickens, effluent pond water and wild ducks on the pond. An isolate from the pond belonged to the same ET as one from a pig, whereas the duck isolates were distinct. This study demonstrates the complex epidemiology of B. pilosicoli infections in piggeries.  相似文献   

20.
Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.  相似文献   

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