Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis.

Introduction: PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens. Methods: Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method. Results: The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/µL for B. hyodysenteriae and L. intracellularis, and 100 copies/µL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively. Discussion: The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

Vaccination with a Lawsonia intracellularis subunit water in oil emulsion vaccine mitigated some disease parameters but failed to affect shedding.

Lawsonia intracellularis is the causative agent of ileitis in swine that manifests as slower weight gain, mild or hemorrhagic diarrhea and/or death in severe cases. As an economically important swine pathogen, development of effective vaccines is important to the swine industry. In developing a subunit vaccine with three recombinant antigens - FliC, GroEL and YopN - we wanted to identify a formulation that would produce robust immune responses that reduce disease parameters associated with Lawsonia intracellularis infection. We formulated these three antigens with four adjuvants: Montanide ISA 660 VG, Montanide Gel 02 PR, Montanide IMS 1313 VG NST, and Montanide ISA 61 VG in an immunogenicity study. Groups vaccinated with formulations including Montanide ISA 660 VG or Montanide ISA 61 VG had significantly more robust immune responses than groups vaccinated with formulations including Montanide Gel 02 PR or Montanide IMS 1313 VG NST. In the challenge study, animals vaccinated with these antigens and Montanide ISA 61 VG had reduced lesion scores, reduced lesion lengths, and increased average daily gain, but no reduction in shedding relative to the control animals. This work shows that this vaccine formulation should be considered for future study in a field and performance trial.

Epidemiology Tools to Evaluate the Control of Proliferative Enteropathy in Commercial Pig Herds.

Proliferative enteropathy (PE) is characterized by diarrhea and reduced weight gains in growing pigs and intestinal hemorrhage in finishers. Vaccination, antibiotic medication, and improved hygiene can control PE, but their efficacy depends upon the epidemiology of PE. This study monitored the timing and severity of PE in 84 commercial pens across seven treatments, including disinfection, vaccination, no treatment, medication with olaquindox (50, 25 and 12.5 ppm), and combined disinfection and vaccination. Vaccination with or without lime disinfection suppressed clinical signs of PE and reduced the number of excreted L. intracellularis relative to untreated pigs housed in cleaned or cleaned and disinfected pens between 9 and 17 weeks of age. Continuous olaquindox mediation to 17 weeks of age prevented L. intracellularis infection, leaving finisher pigs naïve. These finisher pigs suffered an outbreak of hemorrhagic enteropathy with significant reductions in weight gain, feed intake, and mortalities of 4.6%. Over the 13 week grow/finish period, vaccinated pigs housed in disinfected pens showed significantly higher weight gain and feed intake relative to all other treatments, equating to a weight gain difference of between 3.6 and 3.9 kg per pig. Monitoring the immune response and fecal excretion of L. intracellularis in pens of pigs enabled effective PE control strategies to be evaluated on the farm.

Fecal PCR survey and genome analysis of Lawsonia intracellularis in China.

Proliferative enteropathy caused by Lawsonia intracellularis is an important economic associated disease to pig industry, but the knowledge about the prevalence of L. intracellularis in pig farms in China is limited. In addition, there is no complete genome sequence available for L. intracellularis isolates from China. In this study, we developed a TaqMan qPCR for the screening of L. intracellularis by targeting the bacterial 16S rDNA gene. Laboratory evaluations revealed a good sensitivity and specificity on detecting L. intracellularis nucleic acid. Using this method, we investigated 891 fecal samples from apparently healthy pigs in 47 farms. The results demonstrated a screening positive rate of 37.3% (95% CI, 34.1-40.5%) for the samples, and a farm screening positive rate of 93.6% (95% CI, 65.3-94.4%). The screening positive rate at herd level ranged from 6.67% (95% CI, 0.2-31.9%) to 40% (95% CI, 38-79.6%), while at animal level, the highest screening positive rate was found in 12-week-old pigs [85.7% (95% CI, 67.3-96.0%)]. Investigation of 705 diarrheal or bloody feces from symptomatic pigs revealed that the highest positive rate was found in replacement gilts which was 37.18% (95% CI, 45.1-89.5%). Secondly, we conducted the complete genome sequence of a L. intracellularis PPE-GX01-2022 from China through PacBio sequencing. The genome of PPE-GX01-2022 consisted of a chromosome of 1,439,110 bp in length and three plasmids of 193,063, 39,799, and 27,067 bp, respectively. This genome encoded 1,428 predicted proteins, 44 tRNAs, and 6 rRNAs. Sequence comparisons demonstrated that the genome sequence of PPE-GX01-2022 was highly homologous to those of two isolates from US, and these three isolates shared 1,378 core genes. The screening results suggest a high prevalence rate of L. intracellularis in Chinese pig farms. In addition, the genome sequence of the Chinese isolate was highly homologous to those of the field isolates from the US.

Co-Formulation of Recombinant Porcine IL-18 Enhances the Onset of Immune Response in a New Lawsonia intracellularis Vaccine.

Pig is one of the most consumed meats worldwide. One of the main conditions for pig production is Porcine Enteropathy caused by Lawsonia intracellularis. Among the effects of this disease is chronic mild diarrhea, which affects the weight gain of pigs, generating economic losses. Vaccines available to prevent this condition do not have the desired effect, but this limitation can be overcome using adjuvants. Pro-inflammatory cytokines, such as interleukin 18 (IL-18), can improve an immune response, reducing the immune window of protection. In this study, recombinant porcine IL-18 was produced and expressed in Escherichia coli and Pichia pastoris. The protein's biological activity was assessed in vitro and in vivo, and we determined that the P. pastoris protein had better immunostimulatory activity. A vaccine candidate against L. intracellularis, formulated with and without IL-18, was used to determine the pigs' cellular and humoral immune responses. Animals injected with the candidate vaccine co-formulated with IL-18 showed a significant increase of Th1 immune response markers and an earlier increase of antibodies than those vaccinated without the cytokine. This suggests that IL-18 acts as an immunostimulant and vaccine adjuvant to boost the immune response against the antigens, reducing the therapeutic window of recombinant protein-based vaccines.

New Formulation of a Subunit Vaccine Candidate against Lawsonia intracellularis Increases Humoral and Cellular Immune Responses.

Previously, we designed a subunit vaccine candidate based on three L. intracellularis antigens with promising results in pigs. In this study, antigens were produced individually to achieve an even antigen ratio in the formulation. The emulsion characterization included the drop size and the mechanical and thermal stability. Immune response was evaluated by indirect and sandwich ELISAs, qPCR, and flow cytometry. The vaccine candidate's safety was assessed by histopathology and monitoring the clinical behavior of animals. The average production yielded for the chimeric antigen as inclusion bodies was around 75 mg/L. The formulation showed mechanical and thermal stability, with a ratio Hu/Ho > 0.85 and a drop size under 0.15 nm. Antigens formulated at a ratio of 1:1:1 induced a significant immune response in inoculated pigs that persisted until the end of the experiment (week 14). The dose of 200 µg significantly activated cellular response measured by transcriptional and translational levels of cytokines. The cell proliferation assay revealed an increment of lymphocytes T CD4+ at the same dose. Animals gained weight constantly and showed proper clinical behavior during immunization assays. This research demonstrated the immunological robustness of the new subunit vaccine candidate against Porcine Proliferative Enteropathy evenly formulated with three chimeric antigens of L. intracellularis.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis.

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a - Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1-40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Productivity parameters, antimicrobial consumption, and prevalence of enteric pathogens before and after intramuscular vaccination against Lawsonia intracellularis in naturally infected Danish weaner and finisher pig herds.

In Danish pig production, gastro-intestinal diseases account for most of the antimicrobials (AM) used in growing pigs. Diarrhoea is most frequently caused by Lawsonia intracellularis (LI), Brachyspira pilosicoli (BP), E coli fimbria type F4 (F4) and E. coli fimbria type F18 (F18). With a new LI vaccine available from 2019, it was relevant to investigate the effect of this vaccine in a Danish field study including both weaner and finisher sites. The aim was to evaluate the efficacy of Porcilis® Lawsonia Vet. in naturally LI-infected pig herds by comparing of productivity parameters, AM consumption and dynamics of enteric pathogens over two 6-months periods before and after LI vaccination. Further, faecal sock samples were collected from each site before and after vaccination and analysed by qPCR for excretion levels of LI, BP, F18 and F4. In total, 28 weaner and 41 finisher sites were included in the study. Vaccination reduced Feed Conversion Ratio by 0.12 Feed Unit/kg (p = 0.029) and 0.08 Feed Unit/kg (p = 0.005) in weaners and finishers, respectively. Increased Average Daily Weight Gain of 45.6 gr./day (p < 0.001) was found in the finishers. Mortality risk fell by 8.8% in weaners (RR = 0.912; p < 0.001). AM prescriptions for oral group treatments were reduced by 38.8% active compound/kg pig produced (p = 0.005) or 33.3% Weighted Animal Daily Doses per 100 animals per day in finishers (p = 0.004). LI prevalence was reduced in weaners and finishers (both p < 0.001) and BP prevalence was reduced in finishers (p = 0.043). Mean excretion levels of LI and BP decreased at weaner sites (-1.32 and -1.02 log(10) copies/gr faeces, respectively; both p < 0.001) and at finisher sites (-1.04 and -1.16 log(10) copies/gr faeces, respectively; both p < 0.001). Prevalence and excretion levels of F18 and F4 were unaffected by LI vaccination. In conclusion, vaccination against LI using Porcilis® Lawsonia Vet. improved productivity parameters, cut AM consumption, and reduced prevalence and excretion levels of LI and BP in naturally LI-infected pig herds.

Alleviative Effect of Probiotic Ferment on Lawsonia intracellularis Infection in Piglets.

(1) Background: Lawsonia intracellularis (LI) is an obligate intracellular Gram-negative bacterium that causes porcine ileitis. Pigs infected with LI have severe ileal lesions and show symptoms of diarrhea, indigestion, and growth retardation. Previous studies found that probiotic ferment (FAM) improved the growth performance, gut barrier, and function in piglets. Therefore, we aimed to reveal the mechanism that FAM alleviates negative performance in LI-challenged piglets by characterizing the changes in intestinal integrity, function, and gut microbiota following FAM supplementation. (2) Methods: Twenty-four healthy piglets were randomly allotted to four treatments. Three groups were challenged with LI; both FAM addition and vaccination were performed to explore their positive effects on LI-infected piglets. (3) Results: Piglets infected with LI showed lower growth performance and typical pathological symptoms. Moreover, microscopic images showed that observed intestinal morphological damage could be repaired by FAM and vaccine. To explore the digestion of nutrients in piglets, both digestive enzyme activity and ileal transporter expression were performed to reveal the promoting effect of additives. Reduction of LI colonization intervention by FAM could also ameliorate abnormal differentiation and function of intestinal epithelial cells and alleviate severe inflammatory responses in piglets. Regarding the gut microbiota, both the structure and function of the ileal and colonic microbiota were altered following FAM supplementation. (4) Conclusions: In conclusion, probiotic ferment can reduce the colonization of LI in the ileum, improve intestinal damage, barrier function and microbiota structure, and enhance digestive enzyme activity and nutrient transport proteins expression, thereby improving piglet growth performance, which has the effect of preventing ileitis in pigs.

Guar gum modified tilmicosin-loaded sodium alginate/gelatin composite nanogels for effective therapy of porcine proliferative enteritis caused by Lawsonia intracellularis.

In order to overcome the treatment difficulty of Lawsonia intracellularis (L.intracellularis) using antibiotics, the tilmicosin (TIL)-loaded sodium alginate (SA)/gelatin composite nanogels modified with bioadhesive substances were designed. The optimized nanogels were prepared by electrostatic interaction between SA and gelatin at a mass ratio of 1:1 and CaCl2 as an ionic crosslinker and further modified with guar gum (GG). The optimized TIL-nanogels modified with GG had a uniform spherical shape with a diameter of 18.2 ± 0.3 nm, LC of 29.4 ± 0.2 %, EE of 70.4 ± 1.6 %, PDI of 0.30 ± 0.04, and ZP of -32.2 ± 0.5 mv. The FTIR, DSC, and PXRD showed that GG was covered on the surface of TIL-nanogels in a pattern of staggered arrangements. The TIL-nanogels modified with GG had the strongest adhesive strength amongst those with I-carrageenan and locust bean gum and the plain nanogels, and thus significantly enhanced the cellular uptake and accumulation of TIL via clathrin-mediated endocytosis. It exhibited an increased therapeutic effect against L.intracellularis in vitro and in vivo. This study will provide guidance for developing nanogels for intracellular bacterial infection treatment.

Porcine Lawsonia intracellularis Ileitis in Italy and Its Association with Porcine Circovirus Type 2 (PCV2) Infection.

The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease.

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.

Slaughterhouse Visual and Palpation Method for Estimating the Economic Damage of Porcine Proliferative Enteropathy (PPE).

BACKGROUND: Ileitis is a wasting disease of pigs. Clinical symptoms are diarrhea in growing pigs, wasting and reduced performance. Ileitis is ubiquitous in pig producing countries all around the world. It is estimated that the economic losses caused by the disease are USD 4.65 per fattening pig, and American pig farmers lose USD 56.1 million annually. It has become necessary to develop a slaughterhouse ileitis monitoring method that is simple, feasible to perform at modern slaughter lines, leads to immediate results and is cost effective. The practical experiences of applying the method are presented below. METHODS: Our studies were performed on pig herds and slaughterhouses in Central European countries (Hungary, Romania, Poland, Croatia, and Slovakia). Experiences were evaluated based on visual and palpation. The results of our investigations were evaluated by a scoring method. Authors made histological and immunohistochemical examinations of investigated ileums. The hypothetic economic losses due to the disease in each farm were determined by estimating the loss of profit according to Holtkamp's presentation in 2019. RESULTS: The essentials of the method we have developed are: it can be performed during slaughterhouse processing, it does not interfere with or make it impossible to carry out normal technological processes, and the results of the procedure are correlated with the results of other laboratory diagnostic tests for ileitis (histology, immunohistochemistry, herd serology, fecal PCR). It is noteworthy that the results of the method can be used to immediately estimate the impact of Lawsonia intracellularis infection on the performance of the herd from which the slaughter animals come. CONCLUSION: Using the results of the slaughtered pigs' visual and tactile examination at the slaughterhouse, the magnitude of the loss caused by Lawsonia intracellularis infection can be estimated quickly and accurately, and the return on investment of the strategy to be applied can be accurately planned.

Prevalence and risk factors of Brachyspira spp. in pig herds with a history of diarrhoea in six European countries.

Swine dysentery and porcine intestinal spirochaetosis caused by Brachyspira (B.) hyodysenteriae and B. pilosicoli, respectively, are important diseases in swine production worldwide. The aim of this study was to assess the prevalence of both pathogens in farms with a history of diarrhoea within the last 12 months in Denmark, France, Germany, the Netherlands, Spain, and United Kingdom. In addition, risk factors for their prevalence and correlations between presence of different Brachyspira spp. and Lawsonia intracellularis were investigated. Therefore, faecal samples of 6355 nursery to finishing pigs out of 144 herds were sampled in 2017/2018 during a prevalence study on Lawsonia intracellularis, followed by polymerase chain reaction analysis for Brachyspira spp. detection. Herd prevalence differed significantly between countries, from 4.2% to 45.8% for B. hyodysenteriae and 8.3-87.5% for B. pilosicoli, respectively (p < 0.01). For the within-herd prevalence (in positive herds), these values ranged from 2.2% to 27.0% for B. hyodysenteriae and 3.3-50.8% for B. pilosicoli. Mixed infections occurred in 34.1% and 58.7% of B. hyodysenteriae positive samples with Lawsonia intracellularis or B. pilosicoli, respectively. In 43.2% of B. pilosicoli positive samples, Lawsonia intracellularis was detected simultaneously. Overall, nursery pigs were significantly less often positive for one of the pathogens than growing or finishing pigs (p < 0.001). The absence of gastrointestinal problems like diarrhoea, routine use of antimicrobials and well performed biosecurity measures were some of the factors associated with lower detection rate of Brachyspira spp. Surprisingly, deworming of different age categories also showed associations with the detection of Brachyspira spp. which, however, were not always equally directed, and therefore require further investigations. The only risk factor significant for both Brachyspira spp. was the median number of ≥ 30 nursery pigs per pen after weaning, compared to smaller group sizes. Both pathogens were detected with varying frequency between the six European countries. This should be considered in the probability of disease and in case of transnational transport, to prevent spread of pathogens. In addition, the frequent presence of mixed infections in some countries should be taken into account in diagnostics. The most important protective factors against Brachyspira spp. presence on farm are biosecurity measures, while potential new factors such as deworming still require further investigation.

Effect of intramuscular vaccination against Lawsonia intracellularis on production parameters, diarrhea occurrence, antimicrobial treatment, bacterial shedding, and lean meat percentage in two Danish naturally infected finisher pig herds.

Lawsonia intracellularis (LI) is an economically important enteric pathogen in pigs with a worldwide endemic prevalence. The objective of this study was to evaluate the effect of an intramuscularly administrated LI vaccine (Porcilis®Lawsonia Vet.) in Danish finisher pigs (30-115 kg) measured on key production figures, antimicrobial (AB) treatments, occurrence of diarrhea and LI shedding. The study was a group-randomized block-trial with parallel groups in two herds, Herd 1 and Herd 2, experiencing a natural subclinical-clinical LI infection in early finisher period. Vaccination occurred at weaning, but the study focused on the first eight weeks in the finisher period. Further, slaughterhouse data were included. In total, 52 and 50 finisher pens comprising 2184 and 2254 finisher pigs were included in each of two herds, respectively. LI vaccination significantly reduced feed conversion ratio (FCR) by 0.05 and 0.09 FU/kg (p = 0.007 and p < 0.001) alongside a significantly increased average daily weight gain (ADWG) by 31 and 43 gr/day (p = 0.001 and p < 0.001) in each of the herds, respectively. In the vaccinated group, less variation was found in ADWG compared to the control group (p < 0.001 in both herds) as an expression of a more uniform growth, which was further confirmed by less variation in lean meat percent in the vaccinated group in one herd (p = 0.007). No significant difference between groups were found in mortality and pigs excluded due to welfare reasons. AB flock treatment against diarrhea was significantly reduced in Herd 1 with all pens treated in the control group compared to 30.8 % in the vaccinated group (p < 0.001). In Herd 2, the difference was non-significant with 68.0 % in the control group compared to 50.0 % in the vaccination group (p = 0.252). Low levels of individual treatments against diarrhea were seen in both herds (≤ 5.0 %) but still significantly reduced in vaccinated pigs compared to control pigs (p < 0.050 in both herds). Mean diarrheic blot counts were significantly reduced in vaccinated pens compared to control pens (p < 0.001 in both herds). In vaccinated pigs, shedding of LI was reduced in both prevalence (p < 0.001 in both herds), excretion level in positive samples (p < 0.001 in both herds) and, in one herd, also in duration (p = 0.003) when compared to control pigs. In conclusion, pigs vaccinated with Porcilis®Lawsonia Vet against LI in both of two high-health and high-productive finisher herds had, compared to non-vaccinated pigs, significantly improved key production figures, and reduced AB treatment, occurrence of diarrhea, LI shedding, and growth variation.

Infection of juvenile falcons (Falco spp.) with intestinal Lawsonia intracellularis.

Intestinal infection of many host species with Lawsonia intracellularis are widely reported. Analyses of infections among carnivorous falcons have not previously been reported. Fifty juvenile captive falcons (Falco spp.) with or without Lawsonia infection were investigated in the United Arab Emirates, including clinical laboratory methods. Fresh intestinal biopsy samples were analysed by microbiological techniques for Lawsonia and other bacteria and by standard parasitological and pathological methods. Lawsonia intracellularis infection was diagnosed by microbiological examination and qPCR in 10 of 50 juvenile falcons at case examination. Seven of these 10 falcons were of normal clinical appearance, and the other three had other contributing factors to ill-thrift. A range of other conditions were noted in 40 case control falcons. This first report of Lawsonia infection in falcons suggests that the agent may have a limited contribution to clinical disease in these birds, including ill-thrift syndromes. This lack of clinical disease association mimics that noted among Lawsonia infections recorded in other avian families.

Prevalences and excretion levels of Lawsonia intracellularis, Brachyspira pilosicoli and Escherichia coli F4 and F18 in fecal sock samples from Danish weaner and finisher pig batches and the association with diarrhea.

BACKGROUND: Bacterial enteritis in growing pigs is a matter of concern in Danish pig production challenging herd health as well as production economy, and antimicrobial usage. The aim of this observational study using fecal sock samples was to determine the prevalence and excretion level of Lawsonia intracellularis (LI), Brachyspira pilosicoli (BP), Escherichia coli F4 (F4) and F18 (F18) and to investigate associations between prevalence or excretion levels of the bacteria and diarrhea. The study was performed in the late weaner and the early finisher period in herds with a history of diarrhea. Every weaner and finisher herd contributed with one sample each. RESULTS: In total, 47 weaner and 59 finisher herds were sampled. The overall prevalence and excretion levels (median and range in log(10) copies/gram of feces) were for LI 84.0% (median 6.2; range 3.0-7.7), for BP 45.2% (median 5.6; range 3.0-6.6), for F18 20.8% (median 5.7; range 4.7-7.7), and for F4 4.7% (median 5.5; range 5.2-6.0). In both diarrheic and non-diarrheic samples, the most prevalent bacteria were either LI alone or LI and BP in combination. In general, no association was found between increasing total bacterial excretion levels and diarrhea, but prevalence (p = 0.04) and excretion (p < 0.01) level of F18 was found to be significantly higher in diarrheic samples. Further, a significant association was found between low LI excretion level and lack of diarrhea in weaner herds (p = 0.03). A significant positive correlation was found between excretion levels of LI and BP in diarrheic weaner herd samples (p = 0.02). CONCLUSION: Enteric pathogens were prevalent in a wide range of bacterial excretion levels in both diarrheic and non-diarrheic samples. Especially LI and BP were frequently found and with a positive correlation between excretion levels. Even in the absence of diarrhea, high prevalence and excretion levels of LI and BP were detected, thus making the status of diarrhea an insufficient tool for assessing the severity of their infections.

Experimental infection of high health pigs with porcine circovirus type 2 (PCV2) and Lawsonia intracellularis.

Background: Porcine circovirus type 2 (PCV2) and Lawsonia intracellularis infections can cause enteritis in pigs. A Danish study showed a significantly higher probability of detecting PCV2 without concurrent L. intracellularis infection, indicating that one of these pathogens has an impact on the dynamics of the other. Therefore, a delayed co-infection model was set up, initially aiming at investigating the interaction between PCV2 and L. intracellularis in pigs challenged with PCV2 and 2 weeks later with L. intracellularis. But due to PCV2 contamination of the L. intracellularis inoculum the aim was revisited to describing the infection dynamics and pathogenesis of pigs infected with PCV2 followed by delayed simultaneous exposure to PCV2 and L. intracellularis. Twenty-four high-health piglets were divided into three groups of eight pigs (A, B, C) and inoculated at experimental day (EXD) 0 with mock (groups A and B) or PCV2 (group C), and at EXD 14 with mock (group A) or L. intracellularis/PCV2 (groups B and C). The pigs underwent daily clinical examination, and were necropsied at EXD 51-52. Furthermore, histology, immunohistochemistry, serology and PCR for PCV2 and L. intracellularis, and measurement of C-reactive protein were carried out. Results: Group A remained negative for PCV2 and L. intracellularis. Following inoculation with L. intracellularis/PCV2, no significant differences were observed between group B and C, however pigs already infected with PCV2 (group C) showed milder clinical signs and exhibited milder intestinal lesions, less shedding of L. intracellularis and developed higher L. intracellularis antibody titers than the pigs in group B that only received the combined infection. Though the differences between group B and C were non-significant, all results pointed in the same direction, indicating that the pigs in group B were more affected by the L. intracellularis infection compared to the pigs in group C. Conclusions: Previous exposure to PCV2 had limited impact on the subsequent exposure to a combined L. intracellularis/PCV2 inoculation. However, there was a tendency that the infection dynamics of PCV2 and development of antibodies to PCV2 and L. intracellularis were altered in pigs previously exposed to PCV2. These differences should be confirmed in further experimental trials.

The Effectiveness of Commercial Vaccination against Lawsonia intracellularis in Mitigating the Reduction in ADWG, the Increased Mortality and Fecal Shedding of the Vaccinated Pigs: A Systematic Review and Meta-Analysis.

In this study, a systematic review and meta-analysis was conducted to assess the efficacy of commercial vaccines against PPE in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. Of the 373 articles reviewed, 16 fulfilled the pre-specified inclusion criteria. Three independent reviewers extracted the data, and vaccine effectiveness was assessed using the outcomes of interest. The majority of studies had a low or unclear risk of bias as assessed using the ARRIVE guidelines. The results of the meta-analysis indicated that the vaccination resulted in statistically significant reductions in bacterial fecal shedding (odds ratio, OR = 0.122, 95% confidence interval, CI 0.054−0.278) and mortality rate (risk ratio, RR = 0.199; 95% CI, 0.066−0.605). Furthermore, ADWG was significantly increased in the vaccinated pigs compared to the unvaccinated controls (standardized mean difference (SMD) = 0.606, 95% CI 0.243−0.969). In the subgroup analysis, the production phase and study type significantly influenced the effect size (p < 0.1). The Egger's regression test showed no evidence of publication bias (p > 0.1). The effectiveness of commercially available vaccines against PPE-related weight loss, fecal shedding, and mortality suggests that the vaccines may help control PPE on affected swine farms.