Development and validation of a flow cytometry antibody test for Lawsonia intracellularis.

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (106 bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Equine Proliferative Enteropathy in Weanling Foals on A German Breeding Farm: Clinical Course, Treatment and Long-Term Outcome.

The goal of the current report was to describe the clinical signs, therapy and outcome of foals with suspected equine proliferative enteropathy (EPE) due to an infection with Lawsonia intracellularis. Forty foals, born on the same breeding farm, were diagnosed with suspected clinical EPE between September 2019 and January 2020. Data of these cases were analyzed retrospectively regarding the course of the disease, treatment, outcome and long-term prognosis. All horses, including randomly selected control horses, were reassessed about nine months after the suspicion of EPE. The horses affected were between 5 and 10 months of age. Fever was the most common clinical sign. Hypoproteinemia was shown consistently in all cases. Seroconversion was detected in all horses affected, while fecal shedding of Lawsonia intracellularis via qualitative real-time polymerase chain reaction was only found in 21 cases. Treatment was based on tetracyclines and the administration of equine plasma IV. A total of 39 of 40 foals survived EPE. No long-term effects in terms of poor body condition or abnormal blood values were observed. If diagnosed and treated early, EPE can generally be described as a disease with a good prognosis and no long-term effects in Warmblood horses.

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.

Uncommon isolation of Desulfovibrio vulgaris from a depressed fracture wound on the forehead.

Desulfovibrio spp. are gram negative, obligate anaerobes capable of reducing sulfate. They have caused infections in humans, but very rarely. They are slow growers and difficult to identify. Hence, they are often overlooked and their actual presence goes unnoticed. Here, we describe a case of a 15- year old boy who was involved in a road traffic accident and he presented with seropurulent discharge from a depressed fracture wound on the forehead. Desulfovibrio vulgaris (D.vulgaris), was isolated from the pus discharge, the first to be reported. The characteristic desulfoviridin pigment production in the organism aided in the identification. The infection was successfully managed with pain reliever and course of amoxicillin - clavulanic acid and linezolid.

Oral fluid for detection of exposure to Lawsonia intracellularis in naturally infected pigs.

To demonstrate the utility of oral fluid (OF) for indirect diagnostic detection of Lawsonia intracellularis (Li), 15 pig farms were studied. Serum and fecal samples were collected from 20 animals from five different age groups on each farm. OF samples were collected from animals in two pens of the same age groups. Serum and OF samples were analyzed in an immunoperoxidase in monolayer assay (IPMA) for the detection of anti-Li immunoglobulin G (IgG) and A (IgA). Compatible results were found between PCR and IgG in OF in four of the five ages evaluated. Simultaneous detection of IgG in serum and OF was mainly observed on farms showing clinical signs suggestive of porcine proliferative enteropathy (PPE). These findings demonstrate the potential usefulness of OF in detecting anti-Li antibodies as a diagnostic tool that can be used to monitor PPE in herds with clinical signs compatible with the disease.

Rapid and visual detection of Lawsonia intracellularis with an improved recombinase polymerase amplification assay combined with a lateral flow dipstick.

BACKGROUND: Lawsonia intracellularis (L. intracellularis) is the etiologic agent of porcine proliferative enteropathy (PPE), which is reported in many swine breeding countries all over the world, and has caused enormous economic losses in intensive pig production systems. Therefore, the aim of this study was to develop a simple and rapid method for on-site detection of Lawsonia intracellularis (L. intracellularis). As the isothermal recombinase polymerase amplification (RPA) can be performed at a constant temperature and its product is directly observed on a lateral-flow dipstick (LFD) with naked eyes without electrophoresis, the RPA-LFD assay should be useful for field diagnosis of L. intracellularis as well as its detection from clinical samples. RESULTS: The established RPA-LFD assay could be finished in 30 min at a wide temperature range of 25 to 40 °C, and the amplicons could be visualized by naked eyes. The developed RPA-LFD assay was specific to dnaA gene of L. intracellularis, and did not detect nucleic acids extracted from other common gastrointestinal pathogens. The minimum detection of this RPA-LFD method was 400 L. intracellularis per reaction, which was as sensitive as conventional PCR. Further, the RPA-LFD assay was performed with 150 clinical fecal samples and the detection results were compared with conventional PCR. Results showed that the coincidence rate of RPA-LFD and conventional PCR was 100%. CONCLUSIONS: The combined RPA with LFD assay provides a simple, rapid, specific and sensitive alternative for field diagnosis of L. intracellularis infection.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

First report of human infection by Christensenella minuta, a gram-negative, strickly anaerobic rod that inhabits the human intestine.

Christensenella minuta is a Gram-negative strictly anaerobic short rod that inhabits the human gut. This bacterium was isolated in a mixed infection with Desulfovibrio desulfuricansfrom the blood of a patient with a diagnosis of acute appendicitis. The strain was identified by 16S rRNA sequence analysis. As far as we know, this is the first time C.minuta has been isolated from a human clinical specimen.

Clinical utility and performance of sock sampling in weaner pig diarrhoea.

Low pathogen diarrhoea is a group-level diagnosis, characterised by non-haemorrhagic diarrhoea. In the current study, the apparent prevalence of low pathogen diarrhoea outbreaks in Danish herds was investigated along with the clinical utility of a laboratory examination for intestinal disease, agreement between three consecutive herd examinations from the same herd and agreement between quantitative PCR results from pooled faecal samples and sock samples. Twenty-four veterinarians submitted faecal and sock samples for quantitative PCR testing from outbreaks of diarrhoea in nursery pigs (n=38 herds) where the farmer or veterinarian had decided that antimicrobial treatment was necessary. The veterinarians were asked to fill in a questionnaire and participate in telephone interviews. The apparent prevalence of low pathogen diarrhoea was 0.18 (95% CL: 0.08-0.34). Agreement between the veterinarians' clinical aetiological diagnosis and the pooled faecal sample was 0.18 (95% CL: 0.08-0.34), and Cohen's Kappa was 0.03 (95% CL: -0.08 to 0.14). Antibiotic treatment or prevention strategies were changed in 0.63 (95% CL: 0.46-0.78) of the herds, and the veterinarians indicated that, for 0.32 (95% CL: 0.18-0.50) of the herds, changes were related to the diagnostic results from the laboratory examination performed in the study. In 0.16 (95% CL: 0.05-0.36) of the herds, the same infections were demonstrated at all three consecutive examinations. No herds had three consecutive diarrhoea outbreaks classified as low pathogen diarrhoea. For the quantitative results (log10 of the summed amounts of Lawsonia intracellularis, Brachyspira pilosicoli, Escherichia coli F4 and F18) agreement between pooled faecal samples and sock samples was evaluated. Lin's concordance correlation coefficient was 0.69 (95% CL: 0.48-0.82), and the mean difference between the two types of samples was -0.38 log10 bacteria/g faeces (SD=1.59log10 bacteria/g faeces; 95% CI: -0.90 to 0.14log10 bacteria/g faeces). Agreement for the dichotomised results was 0.89 (95% CI: 0.75-0.97) when test results were classified as low pathogen diarrhoea or not, and Cohen's Kappa was 0.61 (95% CI: 0.26-0.95). In relation to detection of the individual infections, agreement was 0.63 (95% CI: 0.46-0.78), and Cohen's Kappa was 0.53 (95% CI: 0.34-0.71). In conclusion, low pathogen diarrhoea is a common finding amongst diarrhoea outbreaks that are subjected to antibiotic batch treatment in Danish nursery pigs. Sock samples seem to offer a reliable diagnostic method with impact on clinical decisions for treatment and prevention. However, both the diarrhoea type and the aetiology change with time in the majority of herds, indicating a potential need for frequent diagnostic examinations.

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 10(7) to 10(8)L. intracellularis, although smaller ADG reductions were also observed (15 g/day) when the number of L. intracellularis increased from 10(6) to 10(7)L. intracellularis.

Lawsonia intracellularis-associated ulcerative and necro-hemorrhagic enteritis in 5 weanling foals.

This report describes 5 cases of fatal Lawsonia intracellularis-associated ulcerative and necro-hemorrhagic enteritis in weanling Thoroughbred and Standardbred foals. The lesions are similar to those of the L. intracellularis-associated ulcerative and necro-hemorrhagic enteritis syndrome in pigs. Two foals had concurrent severe typhlo-colitis as a result of a large burden of encysted cyathostomes. The clinical, diagnostic, and therapeutic challenges, and the potential complications encountered during the management of such cases are discussed.

Entérite ulcérative et nécro-hémorragique associée àLawsonia intracellularischez 5 poulains sevrés. Ce rapport décrit 5 cas mortels d'entérite ulcérative et nécro-hémorragique associée à Lawsonia intracellularis chez des poulains Thoroughbred et Standardbred. Les lésions sont semblables à celles du syndrome de l'entérite ulcérative et nécro-hémorragique associée à L. intracellularis chez les porcs. Deux poulains étaient atteints d'une typhlo-colite grave concomitante en raison d'une charge importante de cyathostomes enkystés. Les difficultés cliniques, diagnostiques et thérapeutiques ainsi que les complications potentielles rencontrées durant la gestion de ces cas sont analysées.(Traduit par Isabelle Vallières).

Lawsonia intracellularis infection and proliferative enteropathy in foals.

Equine proliferative enteropathy (EPE) is a disease of foals caused by the obligate intracellular organism Lawsonia intracellularis. This organism is unique in that it causes proliferation of infected enterocytes, resulting in thickening of the intestinal epithelium, most often the small intestine. This disease affects mainly weanling foals and causes fever, lethargy, peripheral edema, diarrhea, colic and weight loss. The diagnosis of EPE may be challenging and relies on the presence of hypoproteinemia, thickening of segments of the small intestinal wall observed on abdominal ultrasonography, positive serology and molecular detection of L. intracellularis in feces. The epidemiology and genetic basis for pathogenesis for this disease is beginning to be elucidated. Phenotypic traits, genomic features, and gene expression profiles during L. intracellularis infection in vitro and in vivo are presented. In addition, this article reviews the epidemiology, pathological and clinicopathological findings, diagnosis, and control of EPE.

Diagnostic performance of fecal quantitative real-time polymerase chain reaction for detection of Lawsonia intracellularis-associated proliferative enteropathy in nursery pigs.

Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L. intracellularis-associated proliferative enteropathy (PE) under field conditions. Furthermore, the diagnostic performance for different subpopulations of pigs was investigated, including pigs infected or noninfected with Porcine circovirus-2, Brachyspira pilosicoli, and Escherichia coli. The diagnostic performance was evaluated in terms of diagnostic sensitivity and specificity. Data from pigs originating from 20 herds with antibiotic treatment requiring diarrhea outbreaks from a prior study were reused. Before treatment, pigs were randomly selected for histological and immunohistochemical examination of intestinal segments and fecal quantification of L. intracellularis by qPCR. A total of 313 pigs (157 without diarrhea, 156 with diarrhea) were included in the statistical analysis, and 37 pigs (11.8%) were classified as PE positives (defined as proliferative histological lesions in combination with L. intracellularis demonstration by immunohistochemistry). Lawsonia intracellularis was detected by qPCR in feces from 91 pigs (29.1%). A nonparametric receiver operating characteristic (ROC) analysis provided an area under the ROC curve (0.93) and an optimal cutoff value of 4.8 log10 L. intracellularis bacteria/g feces. This cutoff provided a diagnostic sensitivity of 0.84 and diagnostic specificity of 0.93. The diagnostic sensitivity and specificity were significantly different between herds (P < 0.0001). Numerically, diagnostic sensitivity and specificity were different between subpopulations of pigs, but no significant differences were demonstrated.

Within-day repeatability for absolute quantification of Lawsonia intracellularis bacteria in feces from growing pigs.

Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective of the current study was to investigate overall variation within a day for fecal numbers of L. intracellularis bacteria determined by real-time PCR in growing pigs. From each of 30 pigs with an infection of L. intracellularis, 5 fecal samples were collected within 1 day. A total of 150 fecal samples were obtained and subjected to quantitative PCR (qPCR) testing. Mean fecal dry matter content was 14.3% (standard deviation = 4.5%). Two pigs (6.7%) alternated between being L. intracellularis qPCR positive and negative. For 28 pigs, the excreting levels of L. intracellularis were within the dynamic range of the qPCR assay at all sampling points. For these 28 pigs, the mean excretion level of L. intracellularis was 6.1 log(10) bacteria/g feces (standard deviation = 1.2 log(10) bacteria/g feces). The maximum observed difference between 2 fecal samples from the same pig was 1.1 log(10) bacteria/g feces. The average standard deviation for individual pigs was 0.27 log(10) bacteria/g feces. The average coefficient of variation within pigs was 0.04, ranging from 0.006 to 0.08. The results imply that absolute quantification of L. intracellularis by qPCR has acceptable repeatability within 1 day. However, a population-specific proportion of pigs alternating between positive and negative test results must be expected.

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.

Desulfovibrio desulfuricans bacteremia in an immunocompromised host with a liver graft and ulcerative colitis.

Desulfovibrio spp. are anaerobic, sulfate-reducing, nonfermenting, Gram-negative bacteria found in the digestive tract of humans. Identification of these species with conventional methods is difficult. The reported case of a Desulfovibrio desulfuricans bacteremia occurring in an immunocompromised host with ulcerative colitis confirms that this organism may be a possible opportunistic human pathogen.

Serial use of serologic assays and fecal PCR assays to aid in identification of subclinical Lawsonia intracellularis infection for targeted treatment of Thoroughbred foals and weanlings.

OBJECTIVE: To assess the serial use of serum immunoperoxidase monolayer assays (IPMAs) and fecal PCR assays, combined with other diagnostic methods, to identify subclinical Lawsonia intracellularis infections for targeted treatment of Thoroughbred foals and weanlings at farms in which the pathogen was endemic or nonendemic. DESIGN: Evaluation study. ANIMALS: 100 foals and weanlings (53 and 47 at farms in which L intracellularis was endemic and nonendemic, respectively). PROCEDURES: Serum was collected every 4 weeks and tested via IPMA, for antibodies against L intracellularis. Fecal samples were collected every 2 weeks and tested by use of an L intracellularis-specific PCR assay. When results for IPMAs or PCR assays were positive or clinical signs compatible with equine proliferative enteropathy (EPE) were detected, clinicopathologic testing was performed to determine treatment. RESULTS: No foals had positive results for the L intracellularis-specific IPMA until after weaning; 32 of 53 (60.4%) weanlings at the farm in which L intracellularis was endemic and 8 of 47 (170%) at the farm in which L intracellularis was nonendemic had positive IPMA results, whereas the number of weanlings that tested positive via fecal PCR assays at those farms was 6 and 0, respectively. Nineteen of 32 weanlings with positive IPMA results at the farm in which L intracellularis was endemic were treated for EPE; 5 of these had clinical signs of EPE. No weanlings at the nonendemic farm had clinical signs of or were treated for EPE. CONCLUSIONS AND CLINICAL RELEVANCE: IPMA appeared to be a useful means of identifying weanlings exposed to L intracellularis.