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.     

Distribution of Brachyspira hyodysenteriae and Lawsonia intracellularis in healthy and diarrhoeic pigs

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.     

Analysis of bacterial load and prevalence of mixed infections with Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli in German pigs with diarrhoea

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.     

Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.     

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs

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.     

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.     

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

Background

The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed.

Methods

The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum.

Results

The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78^T (ATCC® 27164^T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml.

Conclusions

The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78^T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.     

Brachyspira pilosicoli isolated from pigs in Japan

Two of four weak beta-hemolytic isolates of intestinal spirochetes isolated from pigs in Japan possessed a unique base alignment of TTTTTT on the 16S ribosomal DNA of Brachyspira pilosicoli and were identified as B. pilosicoli. The other two isolates were not identified by this technique. The identified isolates were 4.2 to 11 microm in length and 0.2 to 0.3 microm in diameter, 4 periplasmic flagella at each end were observed dominantly. The isolates were hippurate positive but indole negative. This is the first report on the isolation of B. pilosicoli from pigs in Japan.     

Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not-although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.     

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.     

Detection of Lawsonia intracellularis in the tonsils of pigs with proliferative enteropathy

The presence of Lawsonia intracellularis, the obligate intracellular bacterium causing proliferative enteropathy (PE), in the tonsils of pigs as a locus for infection or extraintestinal occurrence of the bacterium was investigated by PCR and immunohistochemistry. Tonsillar occurrence of L. intracellularis could be part of the pathogenesis of PE and an important risk factor in the spread of the disease. L. intracellularis was detected by only PCR in the tonsils of 2/32 pigs without PE at necropsy but with a clinical history of diarrhoea and detection of the bacterium in faeces 1 to 3 weeks prior to necropsy but not in four pigs with moderate PE lesions. However, L. intracellularis was detected in the tonsils of 4/9 pigs with PE complicated with necroses and in 4/4 pigs with proliferative haemorrhagic enteropathy in which L. intracellularis antigen also was demonstrated in tonsillar macrophages and as intact bacteria in the lumen of the crypts. The results show that L. intracellularis is detectable in the tonsils of pigs and that the tonsillar presence of L. intracellularis appears to be correlated to the severity of the intestinal lesions possibly as a result of local retention and not as part of the pathogenesis of PE.     

The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces

This field study explored the cytokine expression in intestinal tissue and serum from 19 diarrhoeic and 9 healthy pigs in herds with a long-time history of Lawsonia intracellularis-infection. The disease, proliferative enteropathy (PE), is associated with diarrhoea and poor performance in growers and haemorrhagic diarrhoea and sudden death in finisher pigs, but the immunopathology is poorly understood. Histopathology, demonstration of L. intracellularis and porcine circovirus type 2 (PCV2) in intestinal tissue by PCR, and detection of serum antibodies to L. intracellularis, were performed. The presence of IL-1β, IL-6, IL-10, IFN-α, IFN-γ, TNF-α and TGF-β in sera was determined by immunoassays, and intestinal mRNA expression of these cytokines plus IL-12p40 was determined by qPCR. Intestinal specimens from pigs with intestinal adenomatosis (n=2), proliferative haemorrhagic enteropathy or swine dysentery (n=2), and controls (n=2) were analysed by a genome wide porcine microarray. The clinical signs of PE were not always supported by the subsequent analyses, and the presence of PCV2 may have contributed to an increased mRNA expression for IFN-γ in intestinal specimens from some pigs. The limited gene expression in the microarray analyses and the limited expression of cytokines in both sera and intestines, indicate that the immune response is poorly activated in the initial course of an infection with L. intracellularis. However, the gene encoding for insulin-like growth factor binding protein 3 (IGFBP-3) was up-regulated in two pigs with prominent mucosal proliferation.     

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.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

Interactions between dietary fibre, endo-parasites and Lawsonia intracellularis bacteria in grower-finisher pigs

Samples of faeces and feed were collected from grower and finisher pigs kept on 25 commercial breeder-finisher units in the West-Midlands region of England. Faecal samples were examined for parasite eggs (Ascaris suis, Trichuris suum and strongylid species) using faecal flotation; and for Lawsonia intracellularis bacteria using the polymerase chain reaction. Feed samples were subjected to proximate analysis for energy, protein and fibre content and enzymic colorimetry for levels of non-starch polysaccharides (NSPs). Characteristics relating to housing, feeding and dung disposal systems and husbandry practices were recorded for each farm and assessed for their association with the presence of parasites and L. intracellularis at the herd level. Ascaris eggs were identified in 8% of herds, Trichuris eggs in 20% of herds and in strongylid eggs (Oesophogostomum and/or Hyostrongylus) in 44% of herds. Lawsonia intracellularis was detected in 15% of herds investigated. Herds positive for Trichuris and Ascaris had significantly lower levels of digestible energy and higher levels of neutral detergent fibre, total and insoluble NSPs in their diets than negative herds (p < 0.05). Housing weaners on slatted floors was associated with a significant decreased risk of parasite infection in grower-finishers (odds ratio = 0.09, p = 0.04) compared to housing on solid floors. The use of grower diets high in NSPs was associated with an increased risk of Trichuris infection (odds ratio = 27.6, p = 0.007). There was also an association at the herd level between infection with L. intracellularis and the presence of Trichuris eggs (odds ratio = 17.43, p = 0.069). It is concluded that control of dietary fibre intake (NSPs in particular) for growers and environmental hygiene (dung removal) for weaners appear to be the most important factors controlling parasite infection in grower-finisher pigs in the UK at present. The current move towards more straw based systems is thus likely to exacerbate the influence of these factors and is likely to result in increased parasite infection in grower-finisher pigs in the UK.     

Prevalence of exposure and infection of Lawsonia intracellularis among slaughter-age pigs

The extent of clinical or subclinical infection associated with Lawsonia intracellularis within Dutch pig herds was uncertain. A case-control study of slaughter age pigs was used to study natural infection within Dutch herds and to compare diagnostic methods. From six case herds where clinical disease had been identified recently, and six disease-free herds, 40 pigs of slaughter-age were examined postmortem. The diagnostic methods used were: serology, gross examination, Haematoxylin and Eosin stain (HE), Warthin-Starry silver stain, Lawsonia-specific indirect immunoperoxidase of the ileum, and PCR of ileum mucosa and colon contents. There were 59% seropositive pigs in case herds and 26% seropositive pigs in control herds. Using immunohistochemistry, 57% of case herds and 46% of control herds were bacteria positive in the ileum mucosa. It was concluded that a majority of Dutch herds contain L. intracellularis infected finisher pigs. In some herds this is associated with clinical outbreaks of acute haemorrhagic enteropathy but in other herds no clinical disease is apparent. Many seropositive pigs in herds without clinical disease had evidence of Lawsonia antigen in sites other than the apical cytoplasm of proliferating epithelial cells, particularly the supranuclear region. It was uncertain whether to classify these pigs as having "recovered" from an infection or whether they have a sub-clinical or chronic form of the disease. We concluded that PCR examination of faeces and serology probably provide more specific results than gross examinations at slaughter, and that a monoclonal antibody-based examination of ileum mucosa should be the accepted screening method for this infection.     

Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in various animals. The detection of L. intracellularis in clinical and environmental samples is necessary for the diagnosis of infection and epidemiological investigations. For the detection of L. intracellularis in fecal samples, we have developed an immunological method using immunomagnetic separation and ATP bioluminescence. Magnetic beads were coated with an anti-Lawsonia surface antigen (LsaA) antibody in order to capture the L. intracellularis in fecal samples from infected rabbits and the bacteria captured by the immunomagnetic beads were assayed by means of ATP bioluminescence. Our results showed that L. intracelluraris was detected by immunomagnetic separation of bacteria-holding magnetic beads and ATP-based bioluminescence, suggesting that our methods could be useful for the diagnosis of proliferative enteropathy.