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.     

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.     

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.     

猪劳森氏胞内菌TaqMan荧光定量PCR检测方法的建立

为建立一种快速有效的检测猪劳森氏胞内菌(LI)的方法,本研究根据该菌的天冬氨酸氨裂解酶基因保守序列设计合成一对特异性引物和一条TaqMan探针,建立了定量检测LI的荧光定量PCR方法,并对其进行敏感性、特异性、稳定性试验以及与套氏PCR方法的比较试验.结果表明,标准曲线的循环阈值与模板浓度呈现良好的线性关系,相关系数为0.998507;该方法对其他病原体的检测无特异性荧光信号;而且该方法灵敏度高于套式PCR方法.本研究为猪LI的检测提供了一种特异、敏感、快速的定量检测方法.     

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.     

基于胞内劳森菌16S rDNA基因的TaqMan荧光定量PCR检测方法的建立与应用

参考GenBank发表的胞内劳森菌(Lawsonia intracellularis,L.intracellularis)基因组16SrDNA序列,设计1对特异性引物和1条TaqMan探针,以L.intracellularis疫苗核酸为模板,建立L.intracellularis TaqMan实时荧光定量PCR检测方法。对该检测法制作了标准曲线,并进行了特异性、敏感性和重复性试验,并将其应用于猪增生性肠炎诊断。结果表明,所建立的TaqMan实时荧光定量方法特异性强,仅对含有L.intracellularis扩增出S型荧光曲线,而对大肠杆菌、沙门菌、金黄色葡萄球菌、副猪嗜血杆菌等均无扩增;该方法在1.2×102¹.2×108 copies/μL有良好的线性关系,相关系数为0.972;最低检测浓度为10copies/μL,重复性良好。对79份临床样品检测表明,该方法灵敏度高于普通PCR方法,可用于猪增生性肠炎的临床诊断。     

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.     

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.     

猪痢疾短螺旋体荧光PCR检测方法的建立与初步应用

在对设计合成的引物和6-carboxy-fluorescein(6-FAM)荧光素标记的Taq Man水解探针进行筛选的基础上,通过反应体系优化,建立了猪痢疾短螺旋体荧光PCR检测方法。特异性、敏感性、重复性及临床样品的检测结果均表明建立的方法快速、特异、灵敏,整个检测过程(包括样品的前处理时间)可在4 h内完成。建立的方法大大缩短了该病原的检测周期,并为临床诊断提供快速、敏感、特异性强的检测方法。     

胞内劳森菌巢式PCR检测方法的建立及初步临床应用

为了掌握胞内劳森菌在湖北省5个疑似猪增生性肠炎暴发规模化猪场中的流行情况,我们建立了一种从粪便中检测胞内劳森菌的巢式PCR方法,并利用该方法对采集于这5个规模化猪场不同生产阶段不同临床表现猪群的1 003份粪便样品进行监测。结果显示:5个猪场的胞内劳森菌检出率在11.5%~19.4%之间,平均阳性率为15.5%;就临床表现而言,腹泻猪粪便样品中胞内劳森菌的检出率高于非腹泻猪;就生产阶段而言,保育及育肥猪中胞内劳森菌的检出率高于断奶仔猪、母猪和公猪。我们还利用粪便检测胞内劳森菌呈阳性、疑似临床症状明显的猪的肠道制备的感染溶液进行了病例复制,试验猪在灌服感染溶液后出现了与胞内劳森菌感染相似的临床症状及病理变化,并且从感染猪的肠道组织和粪便中检测到了胞内劳森菌的存在。本试验对于进一步了解胞内劳森菌在我国猪群中的流行情况及致病性具有一定的意义。     

胞内劳森菌PCR检测方法的建立及初步应用

为建立一种用于检测胞内劳森菌的检测方法,本试验根据GenBank中公布的胞内劳森菌16SrRNA设计引物建立PCR诊断方法。结果显示:获得了大小为481bp的PCR产物,核苷酸序列同源性分析结果均为99%以上;特异性试验表明该方法对大肠杆菌、沙门菌、志贺菌、猪流行性腹泻病毒和金黄色葡萄球菌均为扩增阴性;敏感性试验表明该方法的最低检出量为32.8μg/L;对20份临床样品阳性检出率为15%。结果表明:该方法具有较好的特异性和敏感性,适用于临床检测胞内劳森菌。     

胞内劳森氏菌PCR检测方法的建立与应用

为建立一种适用于临床检测胞内劳森氏茵(Lawsonia intracellularis,LI)的检测方法,本研究通过比较提取粪便样品基因组DNA的不同方法,结合PCR方法特异性扩增aspA基因检测临床样品中的LI.敏感性试验表明,阳性样本最低检出量为1.7×102 copie/μL;特异性试验表明,该方法对大肠杆菌、沙...     

Porcine proliferative enteropathy in Estonian pig herds: histopathology and detection of Lawsonia intracellularis by PCR

Lawsonia intracellularis is the causative agent of proliferative enteritis in pigs (PPE). This bacterium is difficult to culture from clinical samples and antemortem demonstration is therefore usually performed by PCR on faecal samples. The aim of this study was to elucidate the frequency of L. intracellularis infection in pig herds in Estonia using PCR, histopathological methods and electronmicroscopical studies. The frequency of demonstration of L. intracellularis was highest in 9-12 weeks old pigs (68.1%). It was more frequent in growing pigs with enteritis on small farms where the system of "all-in all-out" was not practiced and where standards of hygiene were poor. Gross and histopathological studies demonstrated that characteristic macroscopic changes associated with PPE were localised to the distal jejunum and ileum.Thickened longitudinal and circumferential folds occurred in the mucosa of the affected regions of the bowel. Samples from pigs aged 4 to 20 weeks exhibited the most intensive inflammatory changes. The distal part of the jejunum, ileum and the upper third of proximal colon and cecum wall were visibly thickened with reduced luminal diameter. Hyperplasia of lymphoid tissue and, in many cases, pseudomembranous or fibrinous inflammation was found. L. intracellularis was detected in 56 young pigs using histopathological methods. Additionally, in 8 of these pigs intracellular bacteria were demonstrated in ilial epithelial cells by transmission electronmicroscopical (TEM) investigation. On the basis of these TEM investigations it was concluded that L. intracellularis causes disturbances of normal growth, differentiation and apoptosis of the epithelial cells of ileum.     

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.     

The prevalences of Brachyspira spp. and Lawsonia intracellularis in Swedish piglet producing herds and wild boar population

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.     

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.     

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.     

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.