A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy

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.     

Evaluation of selective media for primary isolation of Treponema hyodysenteriae and Treponema innocens

A total of 2450 samples of feces, intestinal contents and colon mucosal scrapings were bacteriologically examined. A total of 53 strains of Treponema sp. were isolated, and 45 strains of Bacteroides sp., 30 strains of E. coli, 30 strains of Micrococcus sp. and 10 strains of Streptococcus D isolates were randomly selected. Growth promoting studies showed statistically significant stimulation of Treponema sp. growth by yeast extract, chicken egg yolk and rumen fluid. Different growth inhibitors were also tested. For selective medium the following inhibitors were selected: spectinomycin, colistin, vancomycin, brilliant green. Optimal concentrations of these inhibitors in the medium were determined. Finally TSA medium supplemented with 0.05% yeast extract, 5% bovine blood, 0.01% DTT, 400 micrograms spectinomycin, and 250 micrograms/ml vancomycin, appeared to be optimal selective medium for intestinal Treponema sp. isolation. Quantitative studies showed that the number of Treponema C.F.U. on Songers et al. medium with spectinomycin and on spectinomycin-vancomycin medium, did not differ significantly. The number of overgrowing bacteria was statistically significantly lower on spectinomycin-vancomycin medium, than Songers et al. selective medium with spectinomycin. The TSA supplemented with blood, yeast extract 50 micrograms/ml of colistin and 1 microgram/ml of brilliant green was less selective than spectinomycin-vancomycin medium and inhibited some strains of Treponema sp. In the case of spectinomycin-vancomycin resistant of overgrowing bacteria, colistin-brilliant green medium may be suitable for isolation of Treponema sp.     

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.     

Survival of Brachyspira hyodysenteriae and B. pilosicoli in terrestrial microcosms

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.     

Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces

Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo-rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two-step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 10(3) cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4-5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS.     

Diagnosis of swine dysentery and spirochaetal diarrhea. III. Results of cultural and biochemical differentiation of intestinal Brachyspira species by routine culture from 1997 to 1999

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.     

Detection of Brachyspira hyodysenteriae, Lawsonia intracellularis and Brachyspira pilosicoli in feral pigs

Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact.     

Simultaneous detection of Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in porcine faeces and tissue samples by multiplex-PCR

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.     

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection

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.     

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.     

Prevalence of Brachyspira species isolated from diarrhoeic pigs in Brazil

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.     

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.     

Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tiamulin resistance in porcine Brachyspira pilosicoli isolates

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.     

Luteinizing hormone and growth hormone secretion in ewes infused intracerebroventricularly with neuropeptide Y

Neuropeptide Y (NPY) provides an important hypothalamic link between nutritional status and neuroendocrine mechanisms regulating growth and reproduction. The objective of the following series of experiments was to determine the effects of single or continuous administration of NPY on secretion of luteinizing hormone (LH) and (or) growth hormone (GH). In experiment 1, four ovariectomized (OVX) ewes and four OVX + estrogen-treated ewes each received, in a 4 x 4 Latin Square arrangement of treatments, a single injection of 0, 0.5, 5, or 50 microg NPY via an intracerebroventricular (i.c.v.) cannulae to determine the effects on secretion of GH. NPY significantly elevated serum GH at the 50 microg dose regardless of estrogen exposure (P = 0.003). In experiment 2, eight OVX ewes were infused i.c.v. with NPY or saline (n = 4/trmt) continuously for 20 h in a linearly increasing dose, ending at 50 microg/h NPY. Blood samples were collected via jugular cannulae every 10 min during hour -4-0 (interval 1, pre-treatment), hour 6-10 (interval 2) and hour 16-20 (interval 3) relative to the initiation of infusion (0 h). Mean LH and LH pulse frequency were lower in NPY- versus saline-infused ewes during intervals 2 and 3 (P < 0.01), but NPY had no discernable effect on serum GH (P > 0.10). In experiment 3, four OVX ewes were continuously infused with NPY as in experiment 2, except that the maximum 50 microg/h dose was achieved after only 10 h of infusion. Blood samples were collected every 10 min, beginning 4 h before and continuing until 4h after the NPY infusion. Mean serum LH changed significantly over time (P = 0.0001), decreasing below pre-treatment levels by hour 3 of NPY infusion (P < 0.01), and returning to pre-treatment concentrations following the end of infusion (P > 0.15). Serum GH also changed significantly over time (P < 0.001). Mean GH levels tended to be greater than pre-treatment levels by hour 2 of infusion (P < 0.08), but thereafter returned to basal levels. Serum GH also increased following the end of NPY infusion (P < 0.03). From these data we conclude that NPY exerts a persistent inhibitory effect on secretion of LH, and may stimulate the secretion of GH during the initiation and cessation of infusion of NPY. These observations support a role for NPY in mediating the effects of undernutrition on both LH and GH, and also provide evidence for potential mechanisms by which leptin, acting through NPY, may stimulate the secretion of GH.     

Influence of diet on the experimental infection of pigs with Brachyspira pilosicoli

The effects of five different diets on the experimental infection of pigs with a Danish field isolate of Brachyspira pilosicoli were investigated. The diets tested were a pelleted and a non-pelleted standard diet based on wheat and barley, the standard diet supplemented with 2 per cent lactic acid, a fermented liquid feed and a diet based on cooked rice. Two trials were conducted, each with six groups of six pigs; in each, two of the groups were fed the standard diet. One of these groups and the other four groups were challenged after two weeks on the diets and euthanased four weeks later. The clinical signs of B pilosicoli infection varied from loose stools to watery, mucoid diarrhoea. The group fed the rice diet excreted B pilosicoli in their faeces for a significantly shorter period than the group fed the standard diet (P < 0.01), and fewer of them excreted the organism (P < 0.05). All the pigs fed the pelleted diet excreted B pilosicoli in their faeces, and significantly more of them showed clinical signs of disease than the pigs fed the standard diet (P < 0.05). The fermented liquid feed and the diet containing lactic acid had no significant effect on the excretion of B pilosicoli or on the numbers of pigs showing clinical signs of disease.     

Infections with weakly haemolytic Brachyspira species in pigs with miscellaneous chronic diseases

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.     

Detectability and prevalence of Brachyspira species in herds rearing health class feeder pigs in Finland

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).     

Demonstration of genes encoding virulence and virulence life-style factors in Brachyspira spp. isolates from pigs

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae.     

The use of in-situ hybridization for the detection of Brachyspira spp. in pigs

Diagnosis of Brachyspira infections in swine and the differentiation of the involved bacteria is time-consuming and in most cases unsatisfactory. Detecting Brachyspira directly in the damaged Brachyspira of the large intestine could provide a direct correlation between histological lesionsa and bacterial growth.In this study we investigated whether in-situ hybridization (ISH) with a digoxigenin-labeled RNA-probe is a suitable method for detecting Brachyspira in the mucosa of the large intestine. Formalin-fixed and paraffin-embedded tissue sections of the large intestine from 78 pigs, which showed macroscopic and histological findings of Brachyspira-associated colitis, were stained with hematoxylin and eosin and Warthin-Starry silver impregnation and subjected to ISH. We used a RNA-probe with a length of 334bp, complementary to a part of the 23S rRNA of all members of the genus Brachyspira. All sections were treated with this anti-sense probe and with a sense control probe. 64 samples (82%) showed clearly positive ISH signals. Thus ISH is a suitable method for detecting Brachyspira directly within the lesions of the large intestine. The quantity of Brachyspira identified by ISH was always lower than by Warthin-Starry staining. Whether this reflects lower sensitivity of the ISH technique, or the fact that other bacteria with morphological similarities to Brachyspira were also stained by Warthin-Starry is unknown as yet. The present investigations provide a basis of further research developing specific probes to distinguish between pathogenic and non pathogenic Brachyspira species and probes detecting other bacteria with morphological similarity to Brachyspira.