Lawsonia intracellularis proliferative enteropathy in a foal

A weanling foal was diagnosed with proliferative enteropathy caused by Lawsonia intracellularis based on history, clinical findings of depression, anorexia, weight loss, colic, diarrhea, and ventral edema, and a combination of serology and fecal PCR. An epidemiological investigation on the premises revealed that many of the other foals and adult horses were seropositive for L. intracellularis, despite being clinically normal, and identified a dog as a potential carrier and source of infection for the foal.The foal was successfully treated with a combination of azithromycin and rifampin.     

Lawsonia intracellularis proliferative enteropathy in a filly

Proliferative enteropathy (PE) caused by the obligate intracellular bacterium Lawsonia intracellularis is a disease of high economic impact in swine worldwide. In most other species the disease occurs as a sporadic infection. This paper reports a PE caused by L. intracellularis in a 9-month-old Pura Raza Espa?ola filly with a history of profuse diarrhoea. Pathological lesions consisted of a severe proliferative enteritis associated with argyrophilic bacteria in the apical cytoplasm of proliferating crypt epithelium. Characteristic PCR products confirmed the presumptive diagnosis of L. intracellularis infection. To our knowledge this is the first report of PE in a horse in Europe caused by L. intracellularis.     

Evidence of host adaptation in Lawsonia intracellularis infections

Background

Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.

Materials and methods

Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10⁹L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.

Results

Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.

Conclusions

Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.     

Measurement of the viability of Lawsonia intracellularis

The objective of this study was to develop and test both a flow cytometry method (FCM) and a direct count method (DCM) that both use fluorescent stains to determine the viability of Lawsonia intracellularis (LI), an obligate intracellular bacterium and the cause of proliferative enteropathy (PE) in pigs and other animal species. Live LI were passaged in cell culture and harvested from infected McCoy cells. Dead LI were prepared by exposing live LI to 70% isopropyl alcohol for 30 min. Seven samples with dead:live ratios of 0:100 (live control), 10:90, 30:70, 50:50, 70:30, 90:10, and 100:0 (dead control) were prepared for testing by both the FCM and the DCM. For the FCM, TO-PRO-3 iodine was applied to the samples, and viable LI were counted. For the DCM, the samples were stained with LIVE/DEAD BacLight, which contains SYTO 9 and propidium iodine, then filtered through 0.2-microm Nuclepore black polycarbonate filters, viewed, and counted with the use of an epifluorescence microscope. Data were evaluated by estimating 95% limits of agreement and the concordance correlation coefficient (CCC). The limits of agreement between the FCM and the DCM versus the standard ratio of added LI showed mean differences not equal to zero, suggesting that systematic bias was introduced. The CCC showed almost perfect agreement (r = 0.9898). With a specific fluorescent probe, the FCM is useful and as good as the DCM for determining LI viability.     

Infection dynamics of Lawsonia intracellularis in pig herds

Little information is known about the natural course and within-herd prevalence of porcine proliferative enteropathy caused by Lawsonia intracellularis. The objective of the study was to investigate the within-herd dynamics of naturally acquired L. intracellularis infection in pigs from weaning to slaughter. The study was designed as a longitudinal survey where 100 pigs from five herds were randomly selected at weaning (approximately 4 weeks of age). Every second week until slaughter (10-12 times, i.e. 20-24 weeks) the pigs were weighed and faecal as well as blood samples were collected. Faecal shedding of L. intracellularis was assessed by real time-PCR and sero-conversion by an indirect immunofluorescence antibody test (IFAT). Clinical disease was not reported but infection was present in all herds and the PCR assay indicated infection in 75% of pigs examined. Most L. intracellularis infected pigs were shedding at 10-12 weeks of age (22-29 kg) and shed for 2-6 successive weeks. After 18 weeks of age all shedding had ceased and re-infection at PCR detectable level was not seen. Variable L. intracellularis associated impact on growth rate was observed. Immediately before bacterial shedding and during early infection the average growth rate declined whereas a compensatory impact was observed during later infection and after bacterial shedding had ceased. The performance of the IFAT resembled the bacteriological test almost perfectly. Sero-conversion was first detected at 12-14 weeks of age. Relative to the bacterial shedding, the onset of sero-conversion was a little delayed, in general, most pigs had sero-converted 2 weeks after the first shedding. Once sero-converted, 92% of the pigs remained sero-positive over the entire survey period.     

胞内劳森菌及其所致的增殖性肠病

本综述介绍的胞内劳森菌 ,是一种弯曲状杆菌 ,无鞭毛 ,抗酸染色阳性 ,能为Warthin Starry或Lavaditti镀银法着色。该菌存在于病畜肠细胞原生质内 ,至今不能人工培养 ,但可在一些肠细胞培养中生长 ,引起猪增殖性肠病 ,还见于仓鼠、马、鹿、雪貂、大鼠、兔、狐、鸵鸟、鸸鹋等动物。可用病理学、血清学、细菌学、PCR等方法诊断 ,防治可用抗生素 ,如金霉素、tylosin、dimetridiazole、carbadax、timulin等治疗 ,至今无疫苗。     

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.     

Seroprevalence of Lawsonia intracellularis in large pig production units

In 11 'farrow-to-finish' outdoor or indoor production units, blood samples from late pregnant gilts were tested by indirect immunofluorescence antibody (IFA) serum assay for Lawsonia intracellularis. The offspring of positively tested gilts were tested at 2, 7, 12, 17, 22 and 27 weeks of age for seroprevalence of Lawsonia intracellularis. All offspring of IFA positive gilts were seronegative at 2 and 7 weeks of age. At 12 weeks of age 81.0% of indoor and 51.0% of outdoor pigs were tested positive. While at 17 weeks of age 82.5% of indoor-raised pigs showed seropositivity, in outdoor units the seropositivity declined to 31.3%. At weeks 22 and 27 indoor-raised pigs still showed marked seropositivity (17.7% and 11.5%) but their outdoor-raised counterparts revealed declining values (7.4% and 0%).     

Lawsonia intracellularis infection in horses: 2005-2007

Background: Lawsonia intracellularis is an emerging equine pathogen that is a cause of equine proliferative enteropathy (EPE).
Objective: To describe the signalment, month of presentation, common clinical signs, clinicopathologic values, diagnostic tests used, antimicrobial use, and survival status in horses affected with EPE; to evaluate how affected horses sold at public auction as yearlings; and to determine results of fecal polymerase chain reaction (PCR) and serum immunoperoxidase monolayer assay (IPMA) results in age matched, clinically normal herdmates.
Animals: The study group was 57 horses treated for disease associated with L. intracellularis infection between August 2005 and January 2007.
Methods: Retrospective study examined horses exhibiting evidence of infection with L. intracellularis and testing positive for fecal PCR or serum IPMA.
Results: Horses ranged in age from 2 to 8 months with a median age of 6 months, and all were examined between August and January. Ventral edema was present in 81% of horses and hypoalbuminemia occurred in all horses. Only 50% of horses tested positive on both PCR and IPMA. Ninety-three percent of horses survived, and survival was unrelated to antimicrobial administered. Affected horses sold as yearlings an average of 68% less than other yearlings by the same sire. Age matched, clinically normal herdmates also tested positive for L. intracellularis on fecal PCR (6%) and IPMA (33%).
Conclusion: L. intracellularis infection should be considered in young horses with ventral edema and hypoalbuminemia that are examined between August and January. Both fecal PCR and serum IPMA are needed to help determine disease status. Treated animals usually survive, although they do not sell for as high a price at public auction as other yearlings by the same sire. Age matched, clinically normal herdmates also test positive for L. intracellularis on fecal PCR and serum IPMA.     

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。     

Species-specificity of equine and porcine Lawsonia intracellularis isolates in laboratory animals

Lawsonia intracellularis infection causes proliferative enteropathy (PE) in many mammalian species, with porcine and equine proliferative enteropathy (PPE and EPE) known worldwide. Hamsters are a well-published animal model for PPE infection studies in pigs. There is no laboratory animal model for EPE infection studies and it is not known whether there is species-specificity for equine or porcine isolates of L. intracellularis in animal models. The objective of this study was to determine whether it is possible to generate typical EPE lesions in hamsters after inoculation with an equine strain of L. intracellularis (EPE strain) and whether it is comparatively possible to generate PPE lesions in rabbits after inoculation with a porcine strain of L. intracellularis (PPE strain). In 2 separate trials, 4-week-old and 3-week-old weanling golden Syrian hamsters were challenged with EPE strains and compared to uninfected (both trials) and PPE-infected controls (Trial 2 only). Concurrently, 6 female New Zealand white juvenile rabbits were infected with PPE strain and observed concomitantly to 8 similar rabbits infected with EPE strain for a different experiment. Hamsters and rabbits were observed for 21 to 24 days post-infection (DPI), depending on the experiment. Neither infected species developed clinical signs. The presence of disease was assessed with diagnostic techniques classically used for pigs and horses: immune-peroxidase monolayer assay on sera; quantitative polymerase chain reaction (qPCR) detection of molecular DNA in feces; and hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) on intestinal tissues. Our results showed that EPE-challenged hamsters do not develop infection when compared with PPE controls (IHC, P = 0.009; qPCR, P = 0.0003). Conversely, PPE-challenged rabbits do not develop typical intestinal lesions in comparison to EPE-challenged rabbits, with serological response at 14 DPI being significantly lower (P = 0.0023). In conclusion, PPE and EPE strains appear to have different host-specificities for hamsters and rabbits, respectively.     

The seroprevalence of Lawsonia intracellularis in horses in The Netherlands

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease of weanling foals and affects their growth and development. The prevalence of Lawsonia intracellularis in The Netherlands is not known. The aim of the study was to investigate the seroprevalence of Lawsonia intracellularis in horses in The Netherlands. Blood samples were taken from healthy foals before and after weaning and from healthy yearlings and mature horses on farms throughout The Netherlands. These samples were analysed for the presence of Lawsonia intracellularis-specific antibodies with a blocking ELISA. White blood cell count, packed cell volume, and total protein concentration were also measured in all foals. Information regarding housing, pasture access, and contact with pig manure on the premises was obtained for all animals. The prevalence of Lawsonia intracellularis antibodies in foals increased significantly from 15% before weaning to 23% after weaning (p = 0.019); it was 89% in yearlings and 99% in horses older than 2 years. There was no significant difference in seroprevalence between the pasture-kept and stable-confined adult horses (97% and 100%, respectively), and there was no significant influence of contact with pig manure. None of the sampled animals showed clinical disease. In conclusion, the results suggest that Lawsonia intracellularis is widespread in The Netherlands and that seropositivity is not necessarily associated with clinical problems. The high seroprevalence in adult horses suggests long-term persistence of antibodies against Lawsonia intracellularis or constant exposure to the bacterium.     

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.     

Lawsonia intracellularis proliferative enteropathy in a weanling foal in Australia

A 6-month-old Quarter Horse weanling filly was presented with lethargy, weight loss, inappetance, mild diarrhoea, marked ventral oedema and severe panhypoproteinaemia. Serum antibody titres for Lawsonia intracellularis were very high but PCR to detect faecal shedding of the organism was negative. Proliferative enteropathy due to L. intracellularis infection was diagnosed. After treatment for 4 weeks with oral erythromycin and rifampicin the filly made a complete recovery.     

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...     

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.