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.     

Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model

Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5–6‐week‐old) does were infected with 3.0 × 10⁸ L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham‐treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE‐affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE‐lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.     

Comparative genome sequencing identifies a prophage-associated genomic island linked to host adaptation of Lawsonia intracellularis infections

Lawsonia intracellularis is an obligate intracellular bacterium and the causative agent of proliferative enteropathy (PE). The disease is endemic in pigs, emerging in horses and has also been reported in a variety of other animal species, including nonhuman primates. Comparing the whole genome sequences of a homologous porcine L. intracellularis isolate cultivated for 10 and 60 passages in vitro, we identified a 18-kb prophage-associated genomic island in the passage 10 (pathogenic variant) that was lost in the passage 60 (non-pathogenic variant). This chromosomal island comprises 15 genes downstream from the prophage DLP12 integrase gene. The prevalence of this genetic element was evaluated in 12 other L. intracellularis isolates and in 53 infected animals and was found to be conserved in all porcine isolates cultivated for up to 20 passages and was lost in isolates cultivated for more than 40 passages. Furthermore, the prophage region was also present in 26 fecal samples derived from pigs clinically affected with both acute and chronic forms of the disease. Nevertheless, equine L. intracellularis isolates evaluated did not harbor this genomic island regardless of the passage in vitro. Additionally, fecal samples from 21 clinically affected horses and four wild rabbits trapped in horse farms experiencing PE outbreaks did not show this prophage-associated island. Although the presence of this prophage-associated island was not essential for a virulent L. intracellularis phenotype, this genetic element was porcine isolate-specific and potentially contributed to the ecological specialization of this organism for the swine host.     

Transmission of Lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits

The aim of this study was to investigate whether feces from rabbits experimentally infected with Lawsonia intracellularis were infectious to foals. Two rabbits were infected with L. intracellularis, while two rabbits served as controls. Eight foals received daily feces from either the infected or the control rabbits. All rabbits and foals were monitored daily for clinical signs for the entire study period (21 days for rabbits, 42 days for foals). Feces and blood were collected for the PCR detection of L. intracellularis and serologic analysis, respectively. None of the infected rabbits or foals developed clinical signs compatible with proliferative enteropathy. All infected rabbits and foals shed L. intracellularis in their feces and all seroconverted. The results support the role of rabbits as asymptomatic amplifiers of L. intracellularis and their role as sources of infection for susceptible foals.     

Investigation of the Usefulness of Serum Amyloid A in Supporting the Diagnosis of Equine Proliferative Enteropathy

The objective of this study was to determine if serum amyloid A (SAA), a major acute-phase protein, could help support the diagnosis of equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis infection in foals. Archived serum samples from 101 foals with enteric signs and hypoproteinemia were available for SAA testing. Based on immunodiagnostics for L. intracellularis, the foals were divided into EPE-suspect (67) and non–EPE-suspect cases (34). Serum amyloid A values ranged from 0 to 2,761 μg/mL (median 466 μg/mL) and from 0 to 2,555 μg/mL (median 192 μg/mL) for the EPE-suspect and the non–EPE-suspect cases, respectively. Although SAA can be measured patient-side and help determine the severity of the underlying inflammatory condition, SAA was unable to consistently support the diagnosis of EPE in hypoproteinemic foals with enteric signs.     

Oral Infection of Weanling Foals with an Equine Isolate of Lawsonia intracellularis,Agent of Equine Proliferative Enteropathy

Background: Equine proliferative enteropathy (EPE) is an emerging disease of weanling foals. Objectives: Describe clinical, hematologic, biochemical, serologic, molecular, and ultrasonographic findings in foals experimentally infected with Lawsonia intracellularis. Animals: Eight foals. Methods: Recently weaned foals were assigned to either the challenge (n = 3), the sentinel (n = 3), or the control (n = 2) group. Foals were experimentally challenged via intragastric inoculation of 3 × 10¹⁰L. intracellularis organisms grown in culture. Each experimentally infected foal was housed with a sentinel foal in order to assess feco‐oral transmission. All foals were monitored daily for the development of clinical abnormalities and were weighed once weekly for the duration of the study (90 days). Abdominal ultrasound examination was performed weekly. Feces were collected every other day for 60 days, then weekly for an additional 30 days for the quantitative molecular detection of L. intracellularis. Blood was collected weekly for hematologic, biochemical, and serologic analysis. Results: Only challenged foals developed transient clinical signs of EPE consisting of anorexia, lethargy, fever, loose feces, and peripheral edema. Two challenged foals developed transient hypoalbuminemia. Fecal shedding of L. intracellularis was first detected in the challenged foals between days 12 and 18 postinoculation and lasted for 7–21 days. Seroconversion was documented in all challenged foals and in 1 sentinel foal. The remaining sentinel and control foals remained unaffected. Conclusions and Clinical Importance: Clinical EPE of variable severity was induced in all foals infected with L. intracellularis. Furthermore, L. intracellularis can be transmitted via the feco‐oral route to susceptible herdmates.     

Serum folate,cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute,chronic or subclinical Lawsonia intracellularis infection

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B₉) and cobalamin (vitamin B₁₂) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.     

Demographic and Environmental Risk Factors for Exposure to Lawsonia intracellularis in Horses in Israel

Equine proliferative enteropathy (EPE) is an enteric disease of foals that is caused by Lawsonia intracellularis. Clinical cases have been reported worldwide; however, data regarding the epidemiology of L. intracellularis in horses are scarce. Thus far, L. intracellularis has not been reported in the Middle East. The objectives of this study were to determine whether the causative agent of EPE exists in horses in Israel and in the Palestinian Authority and to define environmental and demographic risk factors for exposure. Fecal and serum samples were collected from horses from various regions of the country. The presence of L. intracellularis in horses in Israel was determined by real-time polymerase chain reaction (PCR), and seroprevalence was determined by enzyme-linked immunosorbent assay. One fecal sample of 136 tested (0.7%), was PCR positive. Sixty-seven sera samples (30.5%) of 220 horses in sentinel farms had anti-L. intracellularis antibodies. Low seroprevalence was found in foals both from Israel and from the Palestinian Authority (4.2% and 13.3%, respectively). In logistic regression models, geographical locations, management type, and age were found to be significant risk factors associated with seroprevlaence to L. intracellularis. No significant correlation was found between environmental variables and L. intracellularis seroprevalence after controlling for management type. These results support the existence of L. intracellularis in horses in both Israel and the Palestinian Authority. The reasons for the relatively low prevalence are currently not known and may be the result of different management, low exposure to free-living animals, and differences in environmental variables affecting the bacterial burden.     

Comparison of different methods for diagnosis of porcine proliferative enteropathy

The objectives of this study were: (1) to compare 2 methods of serology; (2) to compare 3 histologic techniques; and (3) to compare 2 methods of detecting shedding in pigs experimentally challenged with Lawsonia intracellularis. The sensitivities of these tests were determined by the detection of infection. Forty 5-week-old pigs were inoculated on day 0 with intestinal homogenate from pigs with proliferative enteropathy (PE). Clinical evaluation was done on day 7 and daily from day 14 to 28 postinoculation. Fecal shedding of L. intracellularis was monitored by use of polymerase chain reaction (PCR) analysis and immunoperoxidase staining at 7-day intervals. Serum was obtained on days 0 and 28 for serologic testing by glass slide and tissue culture indirect fluorescent antibody tests. At euthanasia on day 28, gross intestinal lesions were evaluated and ileum samples collected for histologic analyses. Ileal histologic sections from each animal were stained by hematoxylin and eosin, Warthin-Starry silver stain, and immunohistochemistry (IHC). Of the 40 pigs, 36 had gross lesions typical of PE at necropsy. The percentage of agreement between the 2 serologic methods was 94.4%. Immunoperoxidase stain of fecal smears was more sensitive than PCR for detecting fecal shedding, especially on day 21 (89.5% and 60.5%, respectively) and day 28 (59.4% and 37.5%, respectively) post-inoculation. The IHC stain was much more sensitive for detecting infection than the routinely used hematoxylin and eosin and Warthin-Starry silver stains. In conclusion, in experimentally infected pigs, both serologic methods were appropriate techniques for detecting infection. For fecal samples, PCR has low sensitivity. Immunohistochemistry is the best diagnostic tool for formalin-fixed samples.     

Equine proliferative enteropathy – a review of recent developments

Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This emerging disease affects mainly weanling foals and causes fever, lethargy, peripheral oedema, diarrhoea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinaemia, thickening of segments of the small intestinal wall observed upon abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in faeces. Although the clinical entity, diagnostic approach and treatment of EPE are well established and described, the epidemiology for this disease has remained largely unaddressed. This article focuses on new developments in the field of EPE, including epidemiology, pathophysiology, clinical signs, diagnosis, treatment and prevention. The Summary is available in Chinese – see Supporting information.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

The critical threshold of Lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs

Serology indicates that Lawsonia intracellularis infection is widespread in many countries, with most pigs seroconverting before 22 weeks of age. However, the majority of animals appear to be sub-clinically affected, demonstrated by the low reported prevalence of diarrhoea. Production losses caused by sub-clinical proliferative enteropathy (PE) are more difficult to diagnose, indicating the need for a quantitative L. intracellularis assay that correlates well with disease severity. In previous studies, increasing numbers of L. intracellularis in pig faeces, quantified with a real time polymerase chain reaction (qPCR), showed a strong negative correlation with average daily gain (ADG).In this study, the association between faecal L. intracellularis numbers and PE severity was examined in two L. intracellularis experimental challenge trials (n₁ = 32 and n₂ = 95). The number of L. intracellularis shed in individual faeces was determined by qPCR on days 0, 7, 14, 17 and 21 days post challenge, and average daily gain was recorded over the same period. The severity of histopathological lesions of PE was scored at 21 days post challenge. L. intracellularis numbers correlated well with histopathology severity and faecal consistency scores (r = 0.72 and 0.68, respectively), and negatively with ADG (r = ?0.44). Large reductions in ADG (131 g/day) occurred when the number of L. intracellularis shed by experimentally challenged pigs increased from 10⁷ to 10⁸ L. intracellularis, although smaller ADG reductions were also observed (15 g/day) when the number of L. intracellularis increased from 10⁶ to 10⁷ L. intracellularis.     

Equine proliferative enteropathy caused by Lawsonia intracellularis

Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This emerging disease affects mainly weanling foals and causes fever, lethargy, peripheral oedema, diarrhoea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinaemia, thickening of segments of the small intestinal wall observed on abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in faeces. Although the clinical entity, diagnostic work-up and treatment of EPE are well established and described, the epidemiology for this disease has remained largely unaddressed. This article reviews the aetiology, epidemiology, clinical signs, diagnosis, treatment and prevention of EPE.     

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.     

Review of methods for the detection of Lawsonia intracellularis infection in pigs

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis–specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.     

Faecal shedding and serological cross‐sectional study of Lawsonia intracellularis in horses in the state of Minas Gerais,Brazil

Reason for performing the study: Proliferative enteropathy, caused by the intracellular bacterium Lawsonia intracellularis, has been described in horses in Australia, the USA, Canada and European countries but has not been reported in Latin America. The prevalence of the disease in horses worldwide is unknown. Objective: To evaluate the presence of subclinical L. intracellularis infection in horses in the state of Minas Gerais, Brazil. Methods: A longitudinal study using serology and PCR for detecting antibodies (IgG) and shedding of L. intracellularis in faecal samples, respectively, was conducted using a total of 223 horses from 14 different horse farms in Minas Gerais, and from the Veterinary School of UFMG equine herds in Minas Gerais. The immunoperoxidase technique in glass slides was used as the serological test. Results: Twenty‐one horse sera had immunoglobulin G titres of 1:60 and were considered positive. The PCR technique in faeces for L. intracellularis DNA identified 7 horses as faecal shedders. Horses shedding the organism appeared healthy, indicating that subclinical infection of L. intracellularis occurred in the horses. Conclusion: Seropositivity and detection of faecal shedding of L. intracellularis indicates the presence of the agent in the equine population in Minas Gerais. Potential relevance: Results of this study should alert clinicians in countries where proliferative enteropthy in horses has not been reported to consider this disease as a possible cause of enteric disease.     

Search for Lawsonia intracellularis and Bilophila wadsworthia in malabsorption-diseased chickens

Proliferative enteropathy is an important enteric disease caused by Lawsonia intracellularis. A wide range of host species can be infected by the same bacterium, yet the clinico-pathologic features among these hosts remains almost identical. The disease has been recognized regularly among ratites, but not in other avian families, such as galliforms, even though these suffer uncharacterized enteric conditions. Fresh ileum-colon contents were obtained from 228, 3- to 8-week-old chickens with enteric disease, kept at 14 large commercial farms in the southern USA. DNA was extracted from each sample and subjected to polymerase chain reactions (PCR) with primers specific to eubacterial DNA, L. intracellularis, and Bilophila wadsworthia. All chicken samples were positive for eubacterial DNA, 29 chickens (13%) were positive for B. wadsworthia DNA, and none were positive for L. intracellularis DNA. Given the ubiquitous nature of L. intracellularis, we consider it likely that some avian families do not carry the necessary mechanism for L. intracellularis viability. Bilophila wadsworthia appears to be a consistent member of the colonic flora of some host animals. Neither bacterium appears to be associated with malabsorption syndromes in chickens.     

胞内劳森菌人工感染猪的病理学观察

将猪源胞内劳森菌GXNN株在兔体传代,用感染兔肠道黏膜及内容物攻毒试验猪,成功复制出猪增生性肠炎,病猪表现为生长缓慢,腹泻,攻毒后12 d~17 d体温升高,肠系膜淋巴结肿大,回肠黏膜皱褶增厚等;病理组织学观察可见回肠有丰富的集合淋巴小结,回肠腺窝杯状细胞消失,部分腺窝上皮细胞增生,淋巴结内的淋巴小结数目增多。     

Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies to Lawsonia intracellularis in experimentally challenged pigs

Objective The ability of a new commercial ELISA to detect pigs with subclinical proliferative enteropathy (PE) was compared with the traditional indirect fluorescent antibody test (IFAT). Methods Serum samples were selected from pigs with known Lawsonia intracellularis infection status and clinical signs of PE, but the sample population consisted predominantly of pigs subclinically affected by PE. Results Significant association and agreement were shown between the ELISA and IFAT assays. ELISA results correlated well with the duration of L. intracellularis shedding as detected by polymerase chain reaction. Conclusion ELISA can be successfully used to monitor L. intracellularis infection in pigs.