Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1ß, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1ß secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Garcinia dulcis Flower Extract Alters Gut Microbiota and Fecal Metabolomic Profiles of 2K1C Hypertensive Rats.

Garcinia dulcis (GD) extract has been found to have anti-hypertensive properties in animal studies. GD can also alter the colonic microbiota of rats. However, the effects of GD on changes in the gut microbiota and metabolomic profiles of normotensive and hypertensive rats are currently unknown. The purpose of this study was to evaluate changes in the gut microbiota and metabolomic profiles of 2-kidneys-1 clip (2K1C) hypertensive rats after feeding with GD flower extract. Rats were randomly divided into the following 4 groups: sham operation (SO) receiving corn oil (CO) (SO + CO), SO receiving GD (SO + GD), 2K1C receiving corn oil (2K1C + CO) and 2K1C receiving GD (2K1C + GD). Body weight (BW) and systolic blood pressure (SBP) were measured weekly throughout the study. Gut microbiota and fecal metabolites were measured from fresh fecal contents. Alpha diversity results demonstrated a similar microbial richness and diversity between groups. Linear discriminant analysis (LDA) effect size (LEfSe) suggested that GD treatment affected gut microbial community structure in both hypertensive and normotensive rats. Feeding rats with GD caused metabolic alterations that rendered 2K1C + GD rats similar to SO + CO and SO + GD rats. Findings suggest that the impact of GD on gut microbiota and metabolite profiles may be related to its anti-hypertensive properties.

Establishment of molecular diagnostics targeting the 23S ribosomal RNA gene for the detection of Mycoplasma suis infection in Thai domestic pigs.

Mycoplasma (M.) suis is a pathogenic hemotropic Mycoplasma sp. that causes acute hemolytic anemia or chronic infection in pigs. M. suis infection can be diagnosed using several methods, including molecular diagnosis such as conventional PCR (cPCR) and quantitative PCR (qPCR). In these cases, the common target is the 16S rRNA gene; however, this genetic marker cannot distinguish hemoplasma at the species level owing to high sequence identity. Therefore, the 23S rRNA gene has emerged as another target gene. Other than PCR, the loop-mediated isothermal amplification (LAMP) method can be applied for M. suis. The objective of the present study was to establish cPCR, TaqMan qPCR, and LAMP assays in which the 23S rRNA gene is used to detect M. suis infection in Thai domestic pigs. The analytical sensitivity of cPCR was determined as 7.46 × 104 copies/µl of plasmid DNA, whereas those of qPCR and LAMP were 7.46 × 102 copies/µl. There was no cross reaction with other pathogens in any of the assays. To evaluate the diagnostic performance of the assays, they were tested using 173 samples of genomic DNA. The detection percentage of M. suis infection was 24.86% (43/173; 95% CI: 18.61%-31.89%), 28.32% (49/173; 95% CI: 21.75%-35.66%), and 29.48% (51/173; 95% CI: 22.80%-36.88%) using cPCR, qPCR, and LAMP, respectively. Using qPCR as a reference assay, cPCR showed 81.63% sensitivity, 97.58% specificity, and an almost perfect level of agreement (kappa = 0.823). In comparison, LAMP showed 77.55% sensitivity, 89.52% specificity, and a substantial level of agreement (kappa = 0.662). All assays tested here could be applied in veterinary diagnostic laboratories for monitoring porcine health in the herds. Furthermore, the LAMP assay could be used as a screening test in farm practice without the need for any special equipment.

Comparative immune responses after vaccination with the formulated inactivated African horse sickness vaccine serotype 1 between naïve horses and pretreated horses with the live-attenuated African horse sickness vaccine.

Background and Aim: African horse sickness (AHS) is a non-contagious, high mortality, and insect-borne disease caused by a double-stranded RNA virus from the genus Orbivirus. The study aimed to develop inactivated vaccines serotype 1 inactivated AHS vaccine (IAV) and to compare the effect of IAV on antibody responses in young naïve horses and adult horses pre-immunized with live-attenuated AHS virus (AHSV) serotypes 1, 3, and 4 live-attenuated vaccine (LAV). Materials and Methods: A total of 27 horses were vaccinated in two trials. Twelve AHS naïve young horses and 15 adult horses were divided into three groups of 4 and 5 horses each, respectively. Horses in control Group 1 were treated with phosphate-buffered saline. Horses in Group 2 were subcutaneously vaccinated with 2 mL of formulated IAV with 10% Gel 01™ (Seppic, France) on day 0 and horses in Group 3 were subcutaneously vaccinated with 2 mL of IAV on day 0 and a booster on day 28. The IAV vaccine was prepared by isolating the AHSV serotype 1 growing on Vero cells, 10× virus titer was concentrated by ultrafiltration and chemically killed by formalin, using 10% Gel 01™ as an adjuvant. Ethylenediaminetetraacetic acid blood samples were taken for hematology, blood biochemistry, and antibody titers using an immunoperoxidase monolayer assay on 158th day post-vaccination. Results: Vaccination with IAV serotype 1 in adult horses pretreated with LAV increased antibody titers more than in young naïve vaccinated horses. The total leukocyte count and %neutrophils significantly increased, while %lymphocytes and %eosinophils significantly decreased on day 1 after vaccination; no local reactions were observed at the site of injection in any group. All biochemical and electrolyte analyte values were within the normal range after vaccination. Conclusion: The formulation of IAV serotype 1 using Gel 01™ as an adjuvant is safe and induces high antibody titers. This IAV formulation induced a high antibody response in horses without causing local reactions and mild systemic effects. However, AHS naïve horses still required ≥2 vaccinations and an annual booster vaccination to achieve high antibody titers.

Porcine reproductive and respiratory syndrome virus induces tight junction barrier dysfunction and cell death in porcine glandular endometrial epithelial cells.

Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2-6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2-4 dpi. PRRSV type 1 infection also induced dead cells (>4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.

Antimicrobial Susceptibility Profile and Whole-Genome Analysis of a Strong Biofilm-Forming Bacillus Sp. B87 Strain Isolated from Food.

Members of the Bacillus cereus group are considered to be foodborne pathogens commonly associated with diarrheal and emetic gastrointestinal syndromes. Biofilm formation is a major virulence determinant of various pathogenic bacteria, including the B. cereus strains, since it can protect the bacteria against antimicrobial agents and the host immune response. Moreover, a biofilm allows the exchange of genetic material, such as antimicrobial resistance genes, among the different bacterial strains inside the matrix. The aim of the current study was to genotypically and phenotypically characterize Bacillus sp. B87, a strain that was isolated from food and which exhibited strong biofilm-forming capacity. Based on the analysis of the phylogenetic relationship, the isolate was phylogenetically mapped close to Bacillus pacificus. Antimicrobial susceptibility testing revealed that the isolate was resistant to tetracycline and ß-lactam antimicrobial agents, which corresponded with the genotypic characterization using the whole-genome analysis. The genome of Bacillus sp. B87 carried the three-component non-hemolytic enterotoxin (NHE), which is a type of enterotoxin that causes diarrheal symptoms. In addition, the genome also contained several genes that participate in biofilm formation, including the pelDEADAFG operon. These findings expand our understanding of antimicrobial resistance and virulence in Bacillus species based on the link between genotypic and phenotypic characterization.

Evaluation of synbiotics as gut health improvement agents against Shiga toxin-producing Escherichia coli isolated from the pig.

Colibacillosis is one of the major health problems in young piglets resulting in poor health and death caused by Escherichia coli producing F18 pili and Shiga toxin 2e. It is pivotal to reduce colibacillosis in weaned piglets to enhance production performance. In this study, we evaluated synbiotics as the gut health improvement agents in the mouse model challenged with Shiga toxin-producing E. coli (STEC) isolated from piglets. Prebiotic lactulose was formulated with each 5.0 × 106 CFU/mL of Pediococcus acidilactici GB-U15, Lactobacillus plantarum GB-U17, and Lactobacillus plantarum GB 1-3 to produce 3 combinations of synbiotics. A total of 40 three weeks old BALB/c mice were randomly assigned to 4 groups (n = 10): a control group and 3 synbiotics treated groups. Each treatment groups were daily administrated with 5.0 × 106 CFU/mL of one synbiotics for the first week, and every 3 days during the second week. All the mice were challenged with 8.0 × 108 CFU/mL of STEC 5 days after animals began to receive synbiotics. Mice treated with synbiotics based on Pediococcus acidilactici GB-U15 and Lactobacillus plantarum GB-U17 significantly improved daily weight gain compared to mice in other groups. While mice treated with GB-U15 showed better fecal index, no significant differences were observed among groups. Gross lesion and histopathological evaluations showed that mice treated with GB-U15 moderately improved recovery from STEC infection. In conclusion, our results suggest that the synbiotics formulated with lactulose and Pediococcus acidilactici GB-U15 have potential benefits to prevent and improve colibacillosis in weaned piglets.

Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011-2016.

Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines.

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

The dynamics of the piglet gut microbiome during the weaning transition in association with health and nutrition.

BACKGROUND: Understanding the composition of the microbial community and its functional capacity during weaning is important for pig production as bacteria play important roles in the pig's health and growth performance. However, limited information is available regarding the composition and function of the gut microbiome of piglets in early-life. Therefore, we performed 16S rRNA gene and whole metagenome shotgun sequencing of DNA from fecal samples from healthy piglets during weaning to measure microbiome shifts, and to identify the potential contribution of the early-life microbiota in shaping piglet health with a focus on microbial stress responses, carbohydrate and amino acid metabolism. RESULTS: The analysis of 16S rRNA genes and whole metagenome shotgun sequencing revealed significant compositional and functional differences between the fecal microbiome in nursing and weaned piglets. The fecal microbiome of the nursing piglets showed higher relative abundance of bacteria in the genus Bacteroides with abundant gene families related to the utilization of lactose and galactose. Prevotella and Lactobacillus were enriched in weaned piglets with an enrichment for the gene families associated with carbohydrate and amino acid metabolism. In addition, an analysis of the functional capacity of the fecal microbiome showed higher abundances of genes associated with heat shock and oxidative stress in the metagenome of weaned piglets compared to nursing piglets. CONCLUSIONS: Overall, our data show that microbial shifts and changes in functional capacities of the piglet fecal microbiome resulted in potential reductions in the effects of stress, including dietary changes that occur during weaning. These results provide us with new insights into the piglet gut microbiome that contributes to the growth of the animal.

Porcine rotavirus C in pigs with gastroenteritis on Thai swine farms, 2011-2016.

Swine are economically important food animals, but highly contagious porcine epidemic diarrhea virus (PEDV) and rotavirus can afflict pig herds and contribute significantly to piglet morbidity and mortality. While there have been studies on rotavirus group A (RVA) in Thailand, reports of rotavirus group C (RVC) are limited. Here, we aimed to identify the prevalence of RVC circulating on Thai commercial swine farms. We analyzed 769 feces and intestine mucosal contents of pigs affected with diarrhea between 2011 and 2016 using RT-PCR specific for the PEDV spike (S), rotavirus glycoprotein (G) VP7, and protease-sensitive protein (P) VP4 genes. We found that 6.6% (51/769) of samples tested positive for RVC, of which 11 samples were co-infected with RVA and four samples were co-infected with PEDV. Three samples tested positive for all three viruses. Phylogenetic analysis of the VP7 gene showed that the most frequent RVC genotype was G1, which grouped with the prototypic RVC Cowden strain. While G6 and G9 were also common, G3 was relatively rare. Analysis of the VP4 gene revealed that the most common P type was P[5], followed by P[4], P[7], and P[1]. In all, there were six G/P combinations (G6P[5], G1P[1], G1P[4], G1P[5], G9P[4], and G9P[7]), of which G6P[5] was the most predominant.

An alternative method for cultivation of Lawsonia intracellularis.

An alternative method for the cultivation of Lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, was developed using an Original Space Bag inflated with a mixture of gas containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen. The flexibility of this protocol allows the testing of various environmental conditions for static cultivation of this bacterium and the development of diagnostic techniques.

Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis.

The objective of the present study was to develop a quantitative polymerase chain reaction (qPCR) assay using SYBR Green for quantification of Lawsonia intracellularis in cell culture and pig fecal samples. Specific primers were designed and tested using the aspartate ammonia-lyase (aspA) gene as a target. Serial 10-fold dilutions of cell culture samples and several sets of spiked feces were used for qPCR optimization. The lower limit of the linear range of the assay in cell culture was 5.1 x 10(2) L. intracellularis/ml. A concentration of between 2.55 x 10(4) and 2.55 x 10(3) L. intracellularis/g was the lower limit of the linear range when testing community DNA from spiked fecal samples. From both cell culture and fecal samples, L. intracellularis could be detected but not accurately quantified at levels approximately 1 log below the linear range. No cross-reactivity of qPCR was found when the assay was tested using the DNA extracted from 16 species of enteric bacteria commonly found in pig feces or closely related to L. intracellularis. The new qPCR assay might prove to be a sensitive, specific, precise, and accurate method for the detection and quantification of L. intracellularis in field samples.

In vitro assessment of the effectiveness of powder disinfectant (Stalosan F) against Lawsonia intracellularis using two different assays.

The objective of this study was to determine the in vitro efficacy of Stalosan F, a mixed chemical and heavy metal disinfectant, against two strains of Lawsonia intracellularis using both a modified tissue culture and a direct count method. For testing as a powder, 1g, 0.5g, or 0.25g of Stalosan F was applied to bacterial solutions spread into sterile dishes. For use as an aqueous suspension, Stalosan F was prepared to final concentrations of 1%, 4%, 8%, 16%, and 32%. In both applications, L. intracellularis was exposed to Stalosan F for 0.5h, 1h, 2h, and 4h. The results showed that both strains were similar in their susceptibilities to Stalosan F. The modified tissue culture assay showed no detectable L. intracellularis in cell culture after exposure to all levels of Stalosan F powder for 0.5h. Furthermore, the number of viable bacteria was markedly reduced in the aqueous concentration of 4% and no L. intracellularis was detected at concentrations of > or =8% for 0.5h. Using the direct count method, detection of live bacteria was less than 1% after exposure to the powder for 0.5h. After exposure to the aqueous form, the number of viable bacteria killed was over 99% in concentrations of > or =16% compared to controls. Our results indicate that Stalosan F in both powder and suspension forms is able to inactivate over 99% of L. intracellularis after 30min of exposure. Furthermore, both laboratory methods can be used to determine the effect of disinfectants on L. intracellularis viability.

In vitro antimicrobial activity against 10 North American and European Lawsonia intracellularis isolates.

The objective of this study was to determine the in vitro minimum inhibitory concentration (MIC) of antimicrobials against 10 isolates of Lawsonia intracellularis, the etiological agent of proliferative enteropathy (PE). Antimicrobials tested included carbadox, chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin. The MIC of each antimicrobial against L. intracellularis was determined using a tissue culture system and was identified as the lowest concentration that inhibited 99% of L. intracellularis growth, as compared to the antimicrobial-free control. Each antimicrobial concentration was evaluated for both intracellular and extracellular activity against L. intracellularis, an obligately intracellular bacterium. When tested for intracellular activity, carbadox, tiamulin, and valnemulin were the most active antimicrobials with MICs of < or =0.5microg/ml. Tylosin (MICs ranging from 0.25 to 32microg/ml) and chlortetracycline (MICs ranging from 0.125 to 64microg/ml) showed intermediate activities and lincomycin (MICs ranging from 8 to >128mIcog/ml) showed the least activity. When tested for extracellular activity, valnemulin (MICs ranging from 0.125 to 4microg/ml) was the most active against most L. intracellularis isolates. Chlortetracycline (MICs ranging from 16 to 64microg/ml), tylosin (MICs ranging from 1 to >128microg/ml), and tiamulin (MICs ranging from 1 to 32microg/ml) showed intermediate activities. Lincomycin (MICs ranging from 32 to >128microg/ml) showed the least activity. Our in vitro results showed that each L. intracellularis isolate had a different antimicrobial sensitivity pattern and these data can be utilized as an in vitro guideline for the further antimicrobial evaluation of field L. intracellularis isolates.

Evaluation of the humoral immune response and fecal shedding in weanling foals following oral and intra-rectal administration of an avirulent live vaccine of Lawsonia intracellularis.

Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis has recently been recognized as an emerging disease in foals. Whilst the clinical entity, diagnostic evaluation and treatment of affected foals have been well established and described, preventive measures for EPE have remained largely unaddressed. The objectives of this study were to investigate the humoral immune response and onset and duration of fecal shedding in foals after oral and intra-rectal administration of a modified-live vaccine of L. intracellularis. Foals were vaccinated twice, 3 weeks apart, via oral drenching after pre-medication with a proton-pump inhibitor (omeprazole; group 1), intra-rectally (group 2) or orally without any pre-medication (group 3). The health status of the foals was monitored daily, with feces and serum collected at regular intervals for Polymerase Chain Reaction (PCR) and serology. All foals remained healthy and no adverse vaccine reactions were observed. Fecal shedding lasted from 1 to 12 days and was mainly detected in foals receiving the intra-rectal vaccine 11-15 days following the first vaccine administration. Serological responses were measured in the majority of the vaccinated foals. All foals vaccinated intra-rectally seroconverted after the first vaccine, compared to 50% and 0% of foals in groups 1 and 3, respectively. Pre-medication with omeprazole prior to oral vaccination in group 1 foals led to an earlier and stronger detectable humoral response compared to non pre-medicated foals.

Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy.

The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.

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.